High throughput screening of gene function using libraries for functional genomics applications

ABSTRACT

Novel means and methods for their use are provided to determine the function of the product(s) of one or more sample nucleic acids. The sample nucleic acids are synthetic oligonucleotides, DNA, or cDNA and encode polypeptides, antisense nucleic acids, or GSEs. The sample nucleic acids are expressed in a host by a vehicle to alter at least one phenotype of the host. The altered phenotype(s) is/are identified as a means to assign a biological function to the product(s) encoded by the sample nucleic acid(s).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 09/358,036,filed on Jul. 21, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/097,239, filed on Jun. 12, 1998, the contents ofthe entirety of both applications are hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to high throughput methods for identifying thefunction of sample nucleic acids and their products. The invention isexemplified by the use of the E1-complementing adenoviral packaging cellline PER.C6 in combination with an E1-deleted plasmid-based generationsystem to produce recombinant adenoviral vectors in a high throughputsetting to functionate the product of a sample nucleic acid.

BACKGROUND

The ultimate goal of the Human Genome Project is to sequence the entirehuman genome. The expected outcome of this effort is a precise map ofthe 70,000–100,000 genes that are expressed in man. However, a fairlycomplete inventory of human coding sequences will most likely bepublicly available sooner. Since the early 1980s, a large number ofExpressed Sequence Tags (ESTs), which are partial DNA sequences readfrom the ends of complementary DNA (cDNA) molecules, have been obtainedby both government and private research organizations. A hallmark ofthese endeavors, carried out by a collaboration between WashingtonUniversity Genome Sequencing Center and members of the IMAGE (IntegratedMolecular Analysis of Gene Expression) consortium(http:/www-bio.llnl.gov/bbrp/image/image.html), has been the rapiddeposition of the sequences into the public domain and the concomitantavailability of the sequence-tagged cDNA clones from severaldistributors (Marra, et al. (1998) Trends Genet. 14 (1):4–7). Atpresent, the collection of cDNAs is believed to represent approximately50,000 different human genes expressed in a variety of tissues includingliver, brain, spleen, B-cells, kidney, muscle, heart, alimentary tract,retina, and hypothalamus, and the number is growing daily.

Recent initiatives like that of the Cancer Genome Anatomy projectsupport an effort to obtain full-length sequences of clones in theUnigene set (a set of cDNA clones that is publicly available) by theyear 1999. At the same time, commercial entities propose to validate40,000 full-length cDNA clones by 1999. These individual clones willthen be available to any interested party. The speed by which the codingsequences of novel genes are identified is in sharp contrast to the rateby which the function of these genes is elucidated. Assigning functionsto the cDNAs in the databases, or functional genomics, is a majorchallenge in biotechnology today.

For decades, novel genes were identified as a result of researchdesigned to explain a biological process or hereditary disease and thefunction of the gene preceded its identification. In functionalgenomics, coding sequences of genes are first cloned and sequenced andthe sequences are then used to find functions. Although other organismssuch as Drosophila, C. elegans, and Zebrafish are highly useful for theanalysis of fundamental genes, animal model systems are inevitable forcomplex mammalian physiological traits (blood glucose, cardiovasculardisease, inflammation). However, the slow rate of reproduction and thehigh housing costs of the animal models are a major limitation to highthroughput functional analysis of genes. Although labor intensiveefforts are made to establish libraries of mouse strains with chemicallyor genetically mutated genes in a search for phenotypes that allow theelucidation of gene function or that are related to human diseases, asystematic analysis of the complete spectrum of mammalian genes, be ithuman or animal, is a significant task.

In order to keep pace with the volume of sequence data, the field offunctional genomics needs the ability to perform high throughputanalysis of true gene function. Recently, a number of techniques havebeen developed that are designed to link tissue and cell specific geneexpression to gene function. These include cDNA microarraying and genechip technology and differential display messenger RNA (mRNA). SerialAnalysis of Gene Expression (SAGE) or differential display of mRNA canidentify genes that are expressed in tumor tissue but are absent in therespective normal or healthy tissue. In this way, potential genes withregulatory functions can be separated from the excess of ubiquitouslyexpressed genes that have a less likely chance to be useful for smalldrug screening or gene therapy projects. Gene chip technology has thepotential to allow the monitoring of gene expression through themeasurement of mRNA expression levels in cells of a large number ofgenes in only a few hours. Cells cultured under a variety of conditionscan be analyzed for their mRNA expression patterns and compared.Currently, DNA microarray chips with 40,000 non-redundant human genesare produced and are planned to be on the market in 1999 (Editorial(1998) Nat. Genet. 18(3):195–7.). However, these techniques areprimarily designed for screening cancer cells and not for screening forspecific gene functions.

Double or triple hybrid systems also are used to add functional data tothe genomic databases. These techniques assay for protein-protein,protein-RNA, or protein-DNA interactions in yeast or mammalian cells(Brent and Finley (1997) Annu. Rev. Genet. 31:663–704). However, thistechnology does not provide a means to assay for a large number of othergene functions such as differentiation, motility, signal transduction,and enzyme and transport activity. Yeast expression systems have beendeveloped which are used to screen for naturally secreted and membraneproteins of mammalian origin (Klein, et al. (1996) Proc. Natl. Acad.Sci. USA 93 (14):7108–13). This system also allows for collapsing oflarge libraries into libraries with certain characteristics that aid inthe identification of specific genes and gene products. One disadvantageof this system is that genes encoding secreted proteins are primarilyselected. A second disadvantage is that the library may be biasedbecause the technology is based on yeast as a heterologous expressionsystem and there will be gene products that are not appropriatelyfolded.

Other current strategies include the creation of transgenic mice orknockout mice. A successful example of gene discovery by such anapproach is the identification of the osteoprotegerin gene. DNAdatabases were screened to select ESTs with features suggesting that thecognate genes encoded secreted proteins. The biological functions of thegenes were assessed by placing the corresponding full-length cDNAs underthe control of a liver-specific promoter. Transgenic mice created witheach of these constructs consequently have high plasma levels of therelevant protein. Subsequently, the transgenic animals were subjected toa battery of qualitative and quantitative phenotypic investigations. Oneof the genes that was transfected into mice produced mice with anincreased bone density, which led subsequently to the discovery of apotent anti-osteoporosis factor (Simonet, et al. (1997) Cell.89(2):309–19). The disadvantages of this method are that the method iscostly and highly time consuming.

The challenge in functional genomics is to develop and refine all theabove-described techniques and integrate their results with existingdata in a well-developed database that provides for the development of apicture of how gene function constitutes cellular metabolism and a meansfor this knowledge to be put to use in the development of novelmedicinal products. The current technologies have limitations and do notnecessarily result in true functional data. Therefore, there is a needfor a method that allows for direct measurement of the function of asingle gene from a collection of genes (gene pools or individual clones)in a high throughput setting in appropriate in vitro assay systems andanimal models.

The development of high throughput screens is discussed in Jayawickremeand Kost, (1997) Curr. Opin. Biotechnol. 8:629–634. A high throughputscreen for rarely transcribed differentially expressed genes isdescribed in von Stein et al., (1997) Nucleic Acids Res. 35: 2598–2602.High throughput genotyping is disclosed in Hall et al., (1996) GenomeRes. 6:781–790. Methods for screening transdominant intracellulareffector peptides and RNA molecules are disclosed in Nolan, WO97/27212and WO/9727213.

DISCLOSURE OF THE INVENTION

The invention includes methods, and compositions for use therein, fordirectly, rapidly, and unambiguously measuring the function of samplenucleic acids of unknown function in a high throughput setting, using aplasmid-based E1-deleted adenoviral vector system and anE1-complementing host cell. The method includes constructing a set ofadapter plasmids by inserting a set of cDNAs, DNAs, ESTs, genes,synthetic oligonucleotides, or a library of nucleic acids intoE1-deleted adapter plasmids; cotransfecting an E1-complementing cellline with the set or library of adapter plasmids and at least oneplasmid having sequences homologous to sequences in the set of adapterplasmids and which also includes all adenoviral genes not provided bythe complementing cell line or adapter plasmids necessary forreplication and packaging to produce a set or library of recombinantadenoviral vectors preferably in a miniaturized, high throughputsetting. To identify and assign a function to product(s) encoded by thesample nucleic acids, a host is transduced in a high throughput settingwith the recombinant adenoviral vectors, which express the product(s) ofthe sample nucleic acids and thereby alter a phenotype of a host. Thealtered phenotype is identified and used as the basis to assign afunction to the product(s) encoded by the sample nucleic acids. Theplasmid-based system is used to rapidly produce adenoviral vectorlibraries that are preferably replications competent adenovirus(“RCA”)-free for high throughput screening. Each step of the method canbe performed in a multiwell format and automated to further increase thecapacity of the system. This high throughput system facilitatesexpression analysis of a large number of sample nucleic acids from humanand other organisms both in vitro and in vivo and is a significantimprovement over other available techniques in the field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Make “HBV poly (A) signal” region in pTN plasmid sequence morevisible by changing border color to black. Support for this revision isfound in the specification on page 40, lines 8 to 9, which describes thepTN plasmid sequence. Change border to black for figure legend as well.

FIG. 2. Close pAT.X/S and pIG.E1a.E1b.X plasmid sequences with a blackline. Plasmids pAT.X/S and pIG.E1a.E1b.X are shown in FIGS. 3A and 4 andFIG. 3B respectively.

FIG. 3A. Make “HBV poly (A) signal” region in pTN and pAT.PCR2.NEOplasmid sequences more visible by changing border colors to black.Support for this revision is found in the specification on page 40,lines 8 to 9, which describes the pTN plasmid sequence. Change border toblack for figure legend as well. See revised figure. Also, the plasmid“pAT PCR2” should include “X/S.” inserted into its name to become“pAT.X/S.PCR2”. Support for this revision is found Page 40, lines 1 to16 of the specification, specifically on lines 7 to 8.

FIG. 3B. Make “HBV poly (A) signal” region in pTN, pAT.PCR2.NEO, pAT.PCR2.NEO.p(A), and pIG.E1A.NEO plasmid sequences more visible bychanging border colors to black. Support for this revision is found inthe specification on page 40, lines 8 to 9, which describes the pTNplasmid sequence. Change border to black for figure legend as well.

FIG. 4. Make “HBV poly (A) signal” region in pIG.E1A.NEO and pIG.E1A.E1Bplasmid sequences more visible by changing border colors to black.Support for this revision is found in the specification on page 40,lines 8 to 9, which describes the pTN plasmid sequence. Change border toblack for figure legend as well.

FIG. 5. Make “HBV poly (A) signal” region in pAT.PCR2.NEO.p(A) plasmidsequence more visible by changing border color to black. Support forthis revision is found in the specification on page 40, lines 8 to 9,which describes the pTN plasmid sequence. Change border to black forfigure legend as well.

FIG. 6: Transformation of primary baby rat kidney (BRK) cells byadenoviral packaging constructs. Subconfluent dishes of BRK cells weretransfected with 1 or 5 μg of either pIG.NEO, pIG.E1A.NEO, pIG.E1A.E1B,pIG.E1A.E1B.X, pAd5XhoIC, or pIG.E1A.NEO plus pDC26, which expresses theAd5 E1B gene under control of the SV40 early promoter. Three weekspost-transfection, foci were visible, cells were fixed, Giemsa stainedand the foci counted. The results shown are the average number of fociper 5 replicate dishes.

FIG. 7: Western blot analysis of A549 clones transfected withpIG.E1A.NEO and human embryonic retinoblasts (HER) cells transfectedwith pIG.E1A.E1B (PER clones). Expression of Ad5 E1A and E1B 55 kDa and21 kDa proteins in transfected A549 cells and PER cells was determinedby Western blot with mouse monoclonal antibodies (Mab) M73, whichrecognizes E1A gene products, and Mabs AIC6 and C1G11, which recognizethe E1B 55 kDa and 21 kDa proteins, respectively. Mab binding wasvisualized using horseradish peroxidase-labelled goat anti-mouseantibody and enhanced chemiluminesence. 293 and 911 cells served ascontrols.

FIG. 8: Southern blot analysis of 293, 911 and PER cell lines. CellularDNA was extracted, HindIII digested, electrophoresed, and transferred toHybond N+ membranes (Amersham). Membranes were hybridized toradiolabelled probes generated by random priming of the SspI-HindIIIfragment of pAd5.SalB (Ad5 nucleotides 342–2805).

FIG. 9: Transfection efficiency of PER.C3, PER.C5, PER.C6 and 911 cells.Cells were cultured in 6-well plates and transfected in duplicate with 5μg pRSV.lacZ by calcium-phosphate co-precipitation. Forty-eight hourspost-transfection, cells were stained with X-Gal and blue cells werecounted. Results shown are the mean percentage of blue cells per well.

FIG. 10: Construction of adenoviral vector, pMLPI.TK. pMLPI.TK wasdesigned to have no sequence overlap with the packaging constructpIG.E1A.E1B. pMLPI.TK was derived from pMLP.TK by deletion of the regionof sequence overlap with pIG.E1A.E1B and deletion of non-codingsequences derived from lacZ. SV40 poly(A) sequences of pMLP.TK were PCRamplified with primers SV40-1 (SEQ ID NO:33), which introduces a BamHIsite, and SV40-2 (SEQ ID NO:34), which introduces a BglII site. pMLP.TKAd5 sequences 2496 to 2779 were PCR amplified with primers Ad5-1 (SEQ IDNO:35), which introduces a BglII site, and Ad5-2 (SEQ ID NO:36). BothPCR products were BglII digested, ligated, and PCR amplified withprimers SV40-1 and Ad5-2. This third PCR product was BamHI and AflIIIdigested and ligated into the corresponding sites of pMLP.TK, producingpMLPI.TK.

FIG. 11A: New adenoviral packaging construct, pIG.E1A.E1B, does not havesequence overlap with new adenoviral vector, pMLPI.TK. Regions ofsequence overlap between the packaging construct pAd5XhoIC, expressed in911 cells, and adenoviral vector pMLP.TK, that can result in homologousrecombination and the formation of RCA are shown. In contrast, there areno regions of sequence overlap between the new packaging constructpIG.E1A.E1B, expressed in PER.C6 cells, and the new adenoviral vectorpMLPI.TK.

FIG. 11B: New adenoviral packaging construct pIG.E1A.NEO, does not havesequence overlap with new adenoviral vector pMLPI.TK. There are noregions of sequence overlap between the new packaging constructpIG.E1A.NEO and the new adenoviral vector pMLPI.TK that can result inhomologous recombination and the formation of RCA.

FIG. 12: Generation of recombinant adenovirus, IG.Ad.MLPI.TK.Recombinant adenovirus IG.Ad.MLPI.TK was generated by co-transfection of293 cells with SalI linearized pMLPI.TK and the right arm of ClaIdigested, wild-type Ad5 DNA. Homologous recombination between linearizedpMLPI.TK and wild-type Ad5 DNA produces IG.Ad.MLPI.TK DNA, whichcontains an E1 deletion of nucleotides 459–3510. 293 cellstranscomplement the deleted Ad5 genome, thereby permitting replicationof the IG.Ad.MLPI.TK DNA and its packaging into virus particles.

FIG. 13: Rationale for the design of adenoviral-derived recombinant DNAmolecules that duplicate and replicate in cells expressing adenoviralreplication proteins. A diagram of the adenoviral double-stranded DNAgenome indicating the approximate locations of E1, E2, E3, E4, and Lregions is shown. The terminal polypeptide (TP) attached to the5′-terminus is indicated by closed circles. The right arm of theadenoviral genome can be purified by removal of the left arm byrestriction enzyme digestion. Following transfection of the right arminto 293 or 911 cells, adenoviral DNA polymerase (white arrow) encodedon the right arm will produce only single-stranded forms. Neither thedouble-stranded or single-stranded DNA can replicate because they lackan inverted terminal repeat (ITR) at one terminus. Providing thesingle-stranded DNA with a sequence that can form a hairpin structure atthe 3′-terminus, which serves as a substrate for DNA polymerase, willextend the hairpin structure along the entire length of the molecule.This molecule can also serve as a substrate for a DNA polymerase, butthe product is a duplicated molecule with ITRs at both termini that canreplicate in the presence of adenoviral proteins.

FIG. 14: Adenoviral genome replication. The adenoviral genome is shownin the top left panel. The origins or replication are located within theleft and right ITRs at the genome ends. DNA replication occurs in twostages. Replication proceeds from one ITR, generating a daughter duplexand a displaced parental single-strand that is coated with adenoviralDNA binding protein (DBP, open circles) and can form a panhandlestructure by annealing of the ITR sequences at both termini. Thepanhandle is a substrate for DNA polymerase (Pol: white arrows) toproduce double-stranded genomic DNA. Alternatively, replication proceedsfrom both ITRs, generating two daughter molecules, thereby obviating therequirement for a panhandle structure.

FIG. 15: Potential hairpin conformation of a single-stranded DNAmolecule that contains the HP/asp sequence (SEQ ID NO:47). Asp718Idigestion of pICLha, containing the cloned oligonucleotides HP/asp1 andHP/asp2, yields a linear double-stranded DNA with an Ad5 ITR at oneterminus and the HP/asp sequence at the other terminus. In cellsexpressing the adenoviral E2 region, a single-stranded DNA is producedwith an Ad5 ITR at the 5′-terminus and the hairpin conformation at the3′-terminus. Once formed, the hairpin can serve as a primer for cellularand/or adenoviral DNA polymerase to convert the single stranded DNA todouble stranded DNA.

FIG. 16: Diagram of pICLhac. pICLhac contains all the elements of pICL(FIG. 19) but also contains the HP/asp sequence in the Asp718 site in anorientation that will produce the hairpin structure shown in FIG. 15,following linearization by Asp718 digestion and transfection into cellsexpressing adenoviral E2 proteins.

FIG. 17: Diagram of pICLhaw. pICLhaw is identical to pICLhac (FIG. 16)except that the inserted HP/asp sequence is in the opposite orientation.

FIG. 18: Schematic representation of pICLI. pICLI contains all theelements of pICL (FIG. 19) but also contains an Ad5 ITR in the Asp718site.

FIG. 19: Diagram of pICL. pICL is derived from the following: (i)nucleotides 1–457, Ad5 nucleotides 1–457 including the left ITR, (ii)nucleotides 458–969, human Cytomegalovirus (CMV) enhancer and immediateearly promoter, (iii) nucleotides 970–1204, SV40 19S exon and truncated16/19S intron, (iv) nucleotides 1218–2987, firefly luciferase gene, (v)nucleotides 3018–3131, SV40 tandem polyadenylation signals from the latetranscript, (vi) nucleotides 3132–5620, pUC 12 sequences including anAsp718 site, and (vii) ampicillin resistance gene in reverseorientation.

FIG. 20: Shows a schematic overview of the adenoviral fragments clonedin pBr322 (plasmid) or pWE15 (cosmid) derived vectors. The top linedepicts the complete adenoviral genome flanked by its ITRs (filledrectangles) and with some restriction sites indicated. Numbers followingrestriction sites indicate approximate digestion sites (in kb) in theAd5 genome.

FIG. 21: Drawing of adapter plasmid pAd/L420-HSA

FIG. 22: Drawing of adapter plasmid pAd/Clip

FIG. 23: Schematic representation of the generation of recombinantadenoviruses using a plasmid-based system. In the top of the figure, thegenome organization of Ad5 is shown with filled boxes representing thedifferent early and late transcription regions and flanking ITRs. Themiddle of the figure represents the two DNAs used for a singlehomologous recombination while the bottom of the figure represents therecombinant virus after transfection into packaging cells.

FIG. 24: Drawing of minimal adenoviral vector pMV/L420H

FIG. 25: Helper construct for replication and packaging of minimaladenoviral vectors. Schematic representation of the cloning steps forthe generation of the helper construct pWE/AdΔ5′.

FIG. 26: Evidence for SV40-LargeT/ori mediated replication of largeadenoviral constructs in COS-1 cells. FIG. 26A shows a schematicrepresentation of construct pWE/Ad.Δ5′. The location of the SV40 orisequence and the fragments used to prepare probes are indicated.Evidence for SV40-LargeT/ori mediated replication of large adenoviralconstructs in COS-1 cells. FIG. 26B shows an autoradiogram of theSouthern blot hybridized to the adenoviral probe. FIG. 26C shows anautoradiogram of the Southern blot hybridized to the pWE probe. Lane 1,marker lane: λ DNA digested with EcoRI and HindIII. Lane 4 is empty.Lanes 2, 5, 7, 9, 11, 13, 15, and 17 contain undigested DNA and Lanes 3,6, 8, 10, 12, 14, 16 and 18 contain MboI digested DNA. All lanes containDNA from COS-1 cells transfected with pWE.pac (lanes 2 and 3),pWE/Ad.Δ5′ construct #1 (lanes 5 and 6), #5 (lanes 7 and 8) and #9(lanes 9 and 10), pWE/Ad.AflII-rITR (lanes 11 and 12), pMV/CMV-LacZ(lanes 13 and 14), pWE.pac digested with PacI (lanes 15 and 16), orpWE/Ad.AflII-rITR digested with PacI (lanes 17 and 18) as described inthe text. Arrows point to the expected positive signal of 1416 bp (FIG.26B) and 887 bp (FIG. 26C).

FIG. 27: Production of E1/E2A deleted adenoviral vectors and itsefficiency in miniaturized PER.C6/E2A based production system (Example10).

FIG. 28: Average titers produced in a 96-well plate as measured using aPER.C6/E2A based plaque assay (Example 11).

FIG. 29: Fidelity of adenoviral vector production miniaturizedPER.C6/E2A based production system for a number of marker and human cDNAtransgenes (Example 12).

FIG. 30: Percentage of wells showing CPE formation after transfection ofPER.C6/E2A cells with pCLIP-LacZ, purified by 6 different protocols(Example 13). Qiagen: standard alkaline lysis followed by Qiagen plasmidpurification; AlkLys: alkaline lysis followed by isopropanolprecipitation, and solubilization in TE buffer; AL+RNAse: alkaline lysisfollowed by isopropanol precipitation, and solubilization in TE buffercontaining RNase at 10 microgram per ml; AL+R+phenol: alkaline lysisfollowed by isopropanol precipitation, and solubilization in TE buffercontaining RNase at 10 microgram per ml, followed by phenol/chloroformextraction and ethanol precipitation; cetyltrimethylammoniumbromide(CTAB): Standard CTAB plasmid isolation; CTAB+phenol: Standard CTABplasmid isolation, but solubilization in TE buffer containing RNase at10 microgram per ml, followed by phenol/chloroform extraction.

FIG. 31: Effect of using digested plasmid for transfection withoutphenol-chloroform extraction (Example 14). The results of allexperiments are depicted and expressed as percentage of wells showingCPE formation. A) LacZ-adapter DNA was isolated using 6 differentisolation methods (see example 13); 1: Qiagen, 2: Alkaline lysis, 3:Alkaline lysis+RNAse treatment, 4: Alkaline lysis+RNAse treatment+p/cpurification of DNA before linerization, 5:cetyltrimethylammoniumbromide (CTAB), 6: CTAB+p/c purification of DNAbefore linerization, rITR was p/c purified, B) Purified and unpurifiedEGFP- and EYFP-adapter DNA, rITR was p/c purified, C) EGFP-adapter DNAand rITR were tested purified and unpurified; 1: Both adapter and rITRpurified (control), 2: rITR purified, adapter DNA unpurified, 3: rITRand adapter unpurified.

FIG. 32: Stability of adenoviral vectors produced in miniaturized formatand incubated for up to three weeks at three different temperatures andmeasured using a plaque forming assay for adenoviral vectors (Example15).

FIG. 33: Efficiencies of virus generation in percentages of CPE aftervirus generation of several adenoviruses (E1 and E2A deleted) containingcDNAs in antisense (AS) orientation (Example 16).

FIGS. 34A–M: Plasmid maps of adenoviral adapter plasmids (Example 17).These adenoviral adapter plasmids are particularly useful for theconstruction of expression libraries in adenoviral vectors such as thesubject of this application. They have very rare restriction sites forthe linearization of adapters and libraries of adapters (with transgenesinserted) and will not inactivate the adapter by digestion of theinserts. In FIG. 34M, the cosmid containing pIPspAdapt5- orpCLIP-IppoI-polynew-derived adenoviral DNA can be used for in vitroligations. Double stranded oligonucleotides containing compatibleoverhangs are ligated between the I-CeuI and PI-SceI sites, betweenI-CeuI and I-PpoI, between I-SceI and PI-SceI, and between I-SceI andI-PpoI. The PacI restriction endonuclease is subsequently used not onlyto linearize the construct after ligation and liberate the left- andright ITRs, but also to eliminate non-recombinants.

FIG. 34N: Percentage of wells showing CPE formation after transfectionof PER.C6/E2A cells with pCLIP-LacZ and the adapter plasmid pIPspAdapt2.

FIG. 35: (Example 19). Percentage of virus producing wells (CPEpositive) in a 96-well plate of PER.C6/E2A cell after propagation of thefreeze/thawed transfected cell lysates. Helper molecules used forcotransfection were (1) pWE/Ad.AflII-rITRsp, (2)pWE/Ad.AflII-rITRsp.dE2A, (3) pWE/Ad.AflII-rITRsp.dXba, and (4)pWE/Ad.AflII-rITR.

FIG. 36 (A and B) (Example 20): Schematic overview of constructing anarrayed adenoviral cDNA expression library.

FIG. 37 (A, B, C, and D) (Example 21): Comparison of cotransfections ofdifferent adapter plasmids and pWE/Ad.AflII-rITRDE2A on 384-well plateswith cotransfections on 96-well plates. The percentage of virusproducing wells (CPE positive wells) scored at different time points asindicated after propagation of the freeze/thawed transfected cell to newPER.C6/E2A cells 5 days after transfection (upper panel) or 7 days aftertransfection (lower panel) is shown.

FIG. 38 (Example 22): The percentage of virus producing wells (CPEpositive wells) scored at different time points as indicated afterchanging the medium of the transfected cells 7 days after transfection(A); after no medium change (B); and after standard propagation of thefreeze/thawed transfected cells to new PER.C6/E2A cells (C).

FIG. 39 (A, B, and C) (Example 23): The percentage of virus producingcells (CPE positive) scored after propagation of the freeze/thawedtransfected cells to new PER.C6/E2A cells, in three differentexperiments using PER.C6/E2A cells for transfections with indicatedconfluency at time of transfection. The figure legend refers to Table 9where the absolute cell numbers from each flask in each experiment werecounted. The cells from these flasks were used to seed 96-well platesfor transfection with adenoviral adapter and helper DNA molecules.

FIG. 40: The use of adenoviral expression vectors as a semi-stableexpression system for assays with a delayed readout of phenotype afterinfection with an adenoviral expression library (Example 24). Transgeneused: Green Fluorescent Protein (EGFP, Clontech). A crude PER.C6/E2Aproduction lysate was used at an multiplicity of infection (MOI) ofabout 500–1000.

FIG. 41: The use of polyethylenimine (PEI) for generating adenoviralvectors in miniaturized format (Example 25). Transfection efficiency,virus formation (CPE), and proliferation (toxicity) are depicted.

FIG. 42: Effect of temperature PEI at time of transfections on CPEefficiency (Example 25). W: Warm (room temperature) and C: Cold (4° C.).

FIG. 43: Effect of PEI transfection volume on transfection efficiencies(Example 25).

FIG. 44: Washing of PER.C6/E2A cells with serum free medium beforeapplying lipofectamine-DNA complex can be omitted (Example 26).

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the invention provides for a library of expressiblenucleic acids comprising a multiplicity of compartments. Eachcompartment comprises at least one vehicle including at least onenucleic acid of the library, whereby the vehicle is capable of veryefficiently introducing at least one nucleic acid into a cell such thatit can be expressed. One advantage of the library is that the librarymay be introduced into cells very efficiently. Another advantage of thelibrary is that it includes a multiplicity of compartments eachincluding at least one nucleic acid. The library may be favorably usedto study the effect of expressed nucleic acid in a cell. A library withthis architecture may be favorably used to rapidly select thosecompartments including at least one nucleic acid which, when expressedin a cell, exerts a certain effect. When a compartment includes only onenucleic acid, then it is known that the nucleic acid exerts the effect.When a compartment includes more than one nucleic acid, it is known thatat least one of the nucleic acids exerts the effect. The advantage ofknowing which compartment includes nucleic acid which can exert acertain effect is greater when the compartment includes relatively fewdifferent nucleic acids and is highest when the compartment includesonly one nucleic acid. It is also advantageous to precisely know thenumber of different nucleic acids per compartment, particularly inlarger libraries.

An expressible nucleic acid may be any expressible nucleic acid such asa nucleic acid coding for a proteinaceous molecule, an RNA molecule, ora DNA molecule.

In a preferred embodiment, the vehicle includes a viral element or afunctional part, derivative and/or analogue thereof. A viral element mayinclude a virus particle such as, but not limited to, an envelopedretrovirus particle or a virus capsid of a non-enveloped virus such as,but not limited to, an adenovirus. A virus particle is favorable sinceit allows the efficient introduction of at least one nucleic acid into acell. A viral element may also include a viral nucleic acid allowing theamplification of the library in cells. A viral element may include aviral nucleic acid allowing the packaging of at least one nucleic acidinto a vehicle, where the vehicle is a virus particle.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid.

A cell may be any kind of cell. Preferably, when the library is screenedfor the presence of nucleic acids with potential therapeutic values, thecell is a eukaryotic cell, especially a mammalian cell.

In one embodiment, at least one compartment includes at least twovehicles. Especially with, but not limited to, large libraries, itbecomes advantageous to reduce the number of compartments to reduce thenumber of screening assays that need to be performed. In such cases,libraries may be provided that include more than one vehicle. If afterscreening, a certain effect is correlated to a certain compartment, thevehicles in the compartment may be analysed separately in an additionalscreening assay to select the vehicle including the nucleic acid theexpression of which exerts the effect. In addition, the presence of morethan one vehicle in a compartment may be advantageous when a librarycontaining one vehicle per compartment is screened for a nucleic acidcapable of exerting an effect in combination with one particular othernucleic acid. The other nucleic acid may then be provided to the cell byadding a vehicle including the particular other nucleic acid to allcompartments prior to performing the screening assay. Similarly, thevehicle may include at least two nucleic acids.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto target the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.

In one embodiment, the nucleic acid derived from an adenovirus includesthe nucleic acid encoding at least one E1-region protein or a functionalpart, derivative, and/or analogue thereof. The presence of theadenoviral nucleic acid encoding an E1-region protein facilitates, atleast in part, replication of the nucleic acid in a cell. Thereplication capacity is favored in certain applications when screeningis done for expressible nucleic acids capable of irradiating tumorcells. In such cases, replication of an adenoviral nucleic acid leadingto the amplification of the vehicle in a mammal including tumor cellsmay lead to the irradiation of metastasised tumor cells. On the otherhand, the presence of an adenoviral nucleic acid encoding an E1-regionprotein may facilitate, at least in part, amplification of the nucleicacid in a cell for the amplification of vehicles including theadenoviral nucleic acid.

In one embodiment, the vehicle further includes a nucleic acid includingan adeno-associated virus terminal repeat or a functional part,derivative, and/or analogue thereof which allows the integration of atleast one nucleic acid in a cell.

In one embodiment, the viral element derived from an adenovirus includesan adenoviral capsid or a functional part, derivative, and/or analoguethereof. Adenovirus biology is also comparatively well known on themolecular level. Many tools for adenoviral vectors have been andcontinue to be developed, thus making an adenoviral capsid a preferredvehicle for incorporating in a library of the invention. An adenovirusis capable of infecting a wide variety of cells. However, differentadenoviral serotypes have different preferences for cells. To combineand widen the target cell population that an adenoviral capsid of theinvention can enter in a preferred embodiment, the vehicle includesadenoviral fiber proteins from at least two adenoviruses.

Another aspect of the invention provides a method for determining atleast one function of at least one nucleic acid present in a libraryaccording to the invention. This method includes transducing amultiplicity of cells with at least one vehicle including at least onenucleic acid from the library, culturing the cells while allowing forexpression of the nucleic acid, and determining the expressed function.Currently, large numbers of nucleic acids are being sequenced andcloned. In fact, cloning and sequencing of nucleic acid proceeds at sucha rate that most of the functions of newly cloned and sequenced nucleicacids are not known. Also, of the nucleic acids with a known function,not all of the functions are known. The current invention provides amethod for determining the function of a nucleic acid. In one aspect,the invention provides a method for screening a library of the inventionin a screening assay wherein a function of a nucleic acid can beassessed. In such an assay, the function is central. A library of theinvention is screened for the presence of expressible nucleic acidscapable of influencing, at least in part, the function.

In a preferred embodiment, the multiplicity of cells is divided over anumber of compartments each including at least one vehicle including atleast one nucleic acid from the library. The number of compartmentspreferably corresponds to the multiplicity of compartments of thelibrary. In a preferred embodiment, the method further includesselecting the vehicle including a desired function.

In another aspect, the invention provides a method for obtaining anexpressible nucleic acid having a desired function when expressed in acell, determining at least one function of at least one nucleic acidpresent in a library according to the invention. The method includestransducing a multiplicity of cells with at least one vehicle includingat least one nucleic acid from the library, culturing the cell whileallowing for expression of at least one nucleic acid, and determiningthe expressed function.

In another aspect, the invention provides a method for producing alibrary including a multiplicity of compartments each including at leastone nucleic acid delivery vehicle and each including at least onenucleic acid. The method includes recombining the vehicle nucleic acidwith at least one nucleic acid, thereby producing a vehicle capable ofdelivering at least one nucleic acid to a cell in an expressible manner.For expression of a nucleic acid, a number of molecular elements wellknown in the field such as, but not limited to, promoters, enhancers,poly-adenylation signals, translation start and stop signals etc., arerequired and/or may be used.

Recombination may be performed through any means such as through meansof molecular cloning and/or polymerase mediated amplification techniquessuch as PCR and NASBA (Organon Teknika). However, recombining preferablyincludes homologous recombination between at least partially overlappingsequences in the vehicle nucleic acid and at least one nucleic acid.Especially for the generation of large viral derived nucleic acids,homologous recombination is preferred. Preferably, the vehicle nucleicacid and/or at least one nucleic acid includes an adenoviral nucleicacid or a functional part, derivative, and/or analogue thereof. In oneexample, the adenoviral nucleic acid includes a host range mutation thatenables the adenovirus to replicate in non-human primate cells.

In one aspect the invention provides a library obtainable by a method ofthe invention.

The invention further provides the use of a library obtainable by amethod of the invention for determining at least one function of atleast one nucleic acid present in a library of the invention.

The invention further provides a method for amplifying a vehicle presentin a library of the invention including providing a cell with thevehicle, culturing the cell, allowing the amplification of the vehicle,and harvesting vehicles amplified by the cell. Preferably, the cell is aprimate cell thereby enabling the amplification of vehicles includingviral elements that allow replication of the vehicle nucleic acid.Preferably, the cell includes a nucleic acid encoding an adenoviralE1-region protein thereby allowing, among other things, theamplification of vehicles including viral elements derived fromadenovirus including adenoviral nucleic acids including a functionaldeletion of at least part of the E1-region. Preferably, the cell is aPER.C6 cell (ECACC deposit number 96022940) or a functional derivativeand/or analogue thereof. A PER.C6 cell (or a functional derivativeand/or analogue thereof) allows the replication of adenoviral nucleicacid with a deletion of the E1-coding region without concomitantproduction of RCA in instances wherein the adenoviral nucleic acid andchromosomal nucleic acid in the PER.C6 cell or functional derivativeand/or analogue thereof do not include sequence overlap that allows forhomologous recombination between the adenoviral and chromosomal nucleicacid leading to the formation of RCA. Preferably, the cell furtherincludes nucleic acid encoding adenovirus E2A and/or an adenoviralE4-region protein or a functional part, derivative, and/or analoguethereof. This allows the replication of adenoviral nucleic acid withfunctional deletions of nucleic acid encoding adenovirus E2A and/or anadenoviral E4-region protein, thereby inhibiting replication of theadenoviral nucleic acid in a cell not including nucleic acid encodingadenovirus E2A and/or an adenoviral E4-region protein or a functionalpart, derivative and/or analogue thereof, for instance a cell capable ofdisplaying a certain function.

In one example, the vehicle nucleic acid does not include sequenceoverlap with other nucleic acids present in the cell, leading to theformation of vehicle nucleic acid capable of replicating in the absenceof E1-region encoded proteins.

The invention further provides a library according to the invention or amethod according to the invention, wherein the multiplicity ofcompartments includes a multiwell format. A multiwell format is verysuited for automated execution of at least part of the methods of theinvention.

In one aspect the invention provides a library wherein at least onenucleic acid encodes a product of unknown fimction.

The library of the invention and/or the methods of the invention arepreferably used or performed in an at least substantially automatedsetting.

The invention further provides a multiplicity of cells including alibrary according to the invention.

The present invention uses high throughput generation of recombinantadenoviral vector libraries containing one or more sample nucleic acids,followed by high throughput screening of the adenoviral vector librariesin a host to alter the phenotype of the host as a means of assigning afunction to expression product(s) of the sample nucleic acids. Librariesof E1-deleted adenoviruses are generated in a high throughput settingusing nucleic acid constructs and transcomplementary packaging cells.The sample nucleic acid libraries can be a set of distinct defined orundefined sequences or can be a pool of undefined or defined sequences.The first nucleic acid construct is a relatively small and easy tomanipulate adapter plasmid containing, in an operable configuration, atleast a left ITR, a packaging signal, and an expression cassette withthe sample nucleic acids. The second nucleic acid construct contains oneor more nucleic acid molecules that partially overlap with each otherand/or with sequences in the first construct. The second construct alsocontains at least all adenovirus sequences necessary for replication andpackaging of a recombinant adenovirus not provided by the adapterplasmid or packaging cells. The second nucleic acid construct is deletedin E1-region sequences and optionally E2B region sequences other thanthose required for virus generation and/or E2A, E3 and/or E4 regionsequences. Cotransfection of the first and second nucleic acidconstructs into the packaging cells leads to homologous recombinationbetween overlapping sequences in the first and second nucleic acidconstructs and among the second nucleic acid constructs when it is madeup of more than one nucleic acid molecule. Generally, the overlappingsequences are no more than 5000 bp and encompass E2B region sequencesessential for virus production. Recombinant viral DNA is generated withan E1-deletion that is able to replicate and propagate in theE1-complementing packaging cells to produce a recombinant adenoviralvector library. The adenoviral vector library is introduced in a highthroughput setting into a host which is grown to allow sufficientexpression of the product(s) encoded by the sample nucleic acids topermit detection and analysis of its biological activity. The host canbe cultured cells in vitro or an animal or plant model. Sufficientexpression of the product(s) encoded by the sample nucleic acids altersthe phenotype of the host. Using any of a variety of in vitro and/or invivo assays for biological activity, the altered phenotype is analyzedand identified and a function is thereby assigned to the product(s) ofthe sample nucleic acids. The plasmid-based adenoviral vector systemsdescribed here provide for the creation of large gene-transfer librariesthat can be used to screen for novel genes applicable to human diseases.Identification of a useful or beneficial biological effect of aparticular adenoviral mediated transduction can result in a useful genetherapeutic product or a target for a small molecule drug for treatmentof human diseases.

There are several advantages to the subject invention over currentlyavailable techniques. The entire process lends itself to automationespecially when implemented in a 96-well or other multi-well format. Thehigh throughput screening, using a number of different in vitro assays,provides a means of efficiently obtaining functional information in arelatively short period of time. The member(s) of the recombinantadenoviral libraries that exhibit or induce a desired phenotype in ahost in vitro or in situ are identified to reduce the libraries to amanageable number of recombinant adenoviral vectors or clones which canbe tested in vitro in an animal model.

Another distinct advantage of the subject invention is that the methodsproduce RCA-free adenoviral libraries. RCA contamination throughout thelibraries could become a major obstacle, especially if libraries arecontinuously amplified for use in multiple screening programs. A furtheradvantage of the subject invention is minimization of the number ofsteps involved in the process. The methods of the subject invention donot require cloning of each individual adenovirus before use in a highthroughput screening program. Other systems include one or more steps inE. coli to achieve homologous recombination for the various adenoviralplasmids necessary for vector generation (Chartier et al, (1996) J.Virol. 70(7):4805–4810; Crouzet et al., (1997) Proc. Natl. Acad. Sci94(4):1414–1419; He et al., (1998) Proc. Natl. Acad. Sci.95(5):2509–2514). Another plasmid system that has been used foradenoviral recombination and adenoviral vector generation, and which isbased on homologous recombination in human cells, is the pBHG series ofplasmids. However, if this plasmid is used in 293 cells, the plasmid canbecome unstable because the plasmid pBHG contains two ITRs closetogether and also can overlap with E1 sequences. All these features areundesirable and lead to RCA production or otherwise erroneous adenoviralvector production (Bett et al., (1994) Proc. Natl. Acad. Sci. USA91(19):8802–8806). The recombinant nucleic acids of the subjectinvention have been designed to avoid constructions with theseundesirable features.

A further advantage of the subject invention is the ability ofrecombinant adenoviruses to efficiently transfer and express recombinantgenes in a variety of mammalian cells and tissues in vitro and in vivo,resulting in the high expression of the transferred sample nucleicacids. The ability to productively infect quiescent cells, furtherexpands the utility of the recombinant adenoviral libraries. Inaddition, high expression levels ensure that the product(s) of thesample nucleic acids will be expressed to sufficient levels to induce achange that can be detected in the phenotype of a host and allow thefunction of the product(s) encoded by the sample nucleic to bedetermined.

The sample nucleic acids can be genomic DNA, cDNA, previously clonedDNA, genes, ESTs, synthetic double stranded oligonucleotides, orrandomized sequences derived from one or multiple related or unrelatedsequences. The sample nucleic acids can also be directly expressed aspolypeptides, antisense nucleic acids, or genetic suppressor elements(GSE). The sample nucleic acid sequences can be obtained from anyorganism including mammals (for example, man, monkey, mouse), fish (forexample, zebrafish, pufferfish, salmon), nematodes (for example, C.elegans), insects (for example, Drosophila), yeasts, fungi, bacteria,and plants. Alternatively, the sample nucleic acids are prepared assynthetic oligonucleotides using commercially available DNA synthesizersand kits. The strand coding the open reading frame of the polypeptide orproduct of the sample nucleic acid and the complementary strand areprepared individually and annealed to form double-stranded DNA. Specialannealing conditions are not required. The sequences of the samplenucleic acids can be randomized or not through mutagenizing ormethodologies promoting recombination. The sample nucleic acids code fora product(s) for which a biological activity is unknown. The phrasebiological activity is intended to mean an activity that is detectableor measurable either in situ, in vivo, or in vitro. Examples of abiological activity include but are not limited to altered viability,morphologic changes, apoptosis induction, DNA synthesis, tumorigenesis,disease or drug susceptibility, chemical responsiveness or secretion,and protein expression.

The sample nucleic acids preferably contain compatible ends tofacilitate ligation to the vector in the correct orientation and tooperatively link the sample nucleic acids to a promoter. For syntheticdouble-stranded oligonucleotide ligation, the ends compatible to thevector can be designed into the oligonucleotides. When the samplenucleic acid is an EST, genomic DNA, cDNA, gene, or previously-clonedDNA, the compatible ends can be formed by restriction enzyme digestionor the ligation of linkers to the DNA containing the appropriaterestriction enzyme sites. Alternatively, the vector can be modified bythe use of linkers. The restriction enzyme sites are chosen so thattranscription of the sample nucleic acid from the vector produces thedesired product, i.e., polypeptide, antisense nucleic acid, or GSE.

The vector into which the sample nucleic acids are preferably introducedcontains, in an operable configuration, an ITR, at least one cloningsite or preferably a multiple cloning site for insertion of a library ofsample nucleic acids, and transcriptional regulatory elements fordelivery and expression of the sample nucleic acids in a host. Itgenerally does not contain E1 region sequences, E2B region sequences(other than those required for late gene expression), E2A regionsequences, E3 region sequences, or E4 region sequences. The E1-deleteddelivery vector or adapter plasmid is digested with the appropriaterestriction enzymes, either simultaneously or sequentially, to producethe appropriate ends for directional cloning of the sample nucleic acidwhether it be synthetic double-stranded oligonucleotides, genomic DNA,cDNA, ESTs, or a previously-cloned DNA. Restriction enzyme digestion isroutinely performed using commercially available reagents according tothe manufacturer's recommendations and varies according to therestriction enzymes chosen. A distinct set or pool of sample nucleicacids is inserted into E1-deleted adapter plasmids to produce acorresponding set or library of plasmids for the production ofadenoviral vectors. An example of an adapter plasmid is pMLPI.TK, whichis made up of adenoviral nucleotides 1–458 followed by the adenoviralmajor late promoter, functionally linked to the herpes simplex virusthymidine kinase gene, and followed by adenoviral nucleotides 3511–6095.Other examples of adapter plasmids are pAd/L420-HSA (FIG. 21) andpAd/Clip (FIG. 22). pAd/L420-HSA contains adenoviral nucleotides 1–454,the L420 promoter linked to the murine HSA gene, a poly-A signal, andadenoviral nucleotides 3511–6095. pAd/CLIP was made from pAd/L420-HSA byreplacement of the expression cassette (L420-HSA) with the CMV promoter,a multiple cloning site, an intron, and a poly-A signal.

Once digested, the vector and sample nucleic acids are purified by gelelectrophoresis. The nucleic acids can be extracted from various gelmatrices by, for example, agarase digestion, electroelution, melting, orhigh salt extraction. In combination with these methods oralternatively, the digested nucleic acids can be purified bychromatography (e.g., Qiagen or equivalent DNA binding resins) orphenol-chloroform extraction followed by ethanol precipitation. Theoptimal purification method depends on the size and type of the vectorand sample nucleic acids. Both can be used without purification.Generally, the sample nucleic acids contain 5′-phosphate groups and thevector is treated with alkaline phosphatase to promote nucleicacid-vector ligation and prevent vector-vector ligation. If the samplenucleic acid is a synthetic oligonucleotide, 5′-phosphate groups areadded by chemical or enzymatic means. For ligation, molar ratios ofsample nucleic acids (insert) to vector DNA range from approximately10:1 to 0.1:1. The ligation reaction is performed using T4 DNA ligase orany other enzyme that catalyzes double-stranded DNA ligation. Reactiontimes and temperature can vary from about 5 minutes to 18 hours, andfrom about 15° to room temperature, respectively.

The magnitude of expression can be modulated using promoters (CMVimmediately early, promoter, SV40 promoter, or retrovirus LTRs) thatdiffer in their transcriptional activity. Operatively linking the samplenucleic acid to a strong promoter such as the CMV immediate earlypromoter and optionally one or more enhancer element(s) results inhigher expression allowing the use of a lower multiplicity of infectionto alter the phenotype of a host. The option of using a lowermultiplicity of infection increases the number of times a library can beused in situ, in vitro, and in vivo. Moreover, the lower themultiplicity of infection and dosages used in screening programs,assays, and animal models decreases the chance of eliciting toxiceffects in the transduced host, which increases the reliability of thesubject of this invention. Another way to reduce possible toxic effectsrelating to expression of the library is to use a regulatable promoter,particularly one which is repressed during virus production but can beturned on or is active during functional screenings with the adenovirallibrary, to provide temporal and/or cell type specific controlthroughout the screening assay. In this way, sample nucleic acids thatare members of the library and are toxic, inhibitory, or in any otherway interfere with adenoviral replication and production, can still beproduced as an adenoviral vector (see International Patent Appl'n WO97/20943). Examples of this type of promoter are the AP1-dependentpromoters which are repressed by adenoviral E1 gene products, resultingin shut off of sample nucleic acid expression during adenoviral libraryproduction (see van Dam et al., (1990) Mol. Cell. Biol.10(11):5857–5864). Such a promoter can be made using combinatorialtechniques or natural or adapted forms of promoters can be included.Examples of AP1-dependent promoters are promoters from the collagenase,c-myc, monocyte chemoattractant protein (JE or mcp-1/JE), andstromelysin genes (Hagmeyer et al., (1993) EMBO J. 12(9);3559–3572;Offringa et al., (1990) Cell 62(23):527–538; Offringa et al., (1988)Nucleic Acids Res. 16(23):10973–10984; van Dam et al., (1989) Oncogene4(10):1207–1212). Alternatively, other more specific but strongerpromoters can be used especially when complex in vitro screenings or invivo models are employed and tissue-regulated expression is desired. Anypromoter/enhancer system functional in the chosen host can be used.Examples of tissue-regulated promoters include promoters with specificactivity or enhanced activity in the liver, such as the albumin promoter(Tronche et al., (1990) Mol. Biol. Med. 7(2):173–185). Another approachto enhanced expression is to increase the half-life of the mRNAtranscribed from the sample nucleic acids by including stabilizingsequences in the 3′ untranslated region. A second nucleic acidconstruct, a helper plasmid having sequences homologous to sequences inthe E1-deleted adapter plasmids, which carries all necessary adenoviralgenes necessary for replication and packaging, also is prepared.Preferably, the helper plasmid has no complementing sequences that areexpressed by the packaging cells that would lead to production of RCA.In addition, preferably the helper plasmids, adapter plasmid, andpackaging cell have no sequence overlap that would lead to homologousrecombination and RCA formation. The region of sequence overlap sharedbetween the adapter plasmid and the helper plasmid allows homologousrecombination and the formation of an E1-deleted, replication-defectiverecombinant adenoviral genome. Generally, the region of overlapencompasses E2B region sequences that are required for late geneexpression. The amount of overlap that provides for the best efficiencywithout appreciably decreasing the size of the library insert can bedetermined empirically. The sequence overlap is generally 10 bp to 5000bp, more preferably 2000 to 3000 bp.

The size of the sample nucleic acids or DNA inserts in a desiredadenoviral library can vary from several hundred base pairs (e.g.,sequences encoding neuropeptides) to more than 30 Kbp (e.g., titin). Thecloning capacity of the adenoviral vectors produced using adapterplasmids can be increased by deletion of additional adenoviral gene(s)that are then easily complemented by a derivative of an E1-complementingcell line. As an example, candidate genes for deletion include E2, E3,and/or E4. For example, regions essential for adenoviral replication andpackaging are deleted from the adapter and helper plasmids andexpressed, for example, by the complementing cell line. For E3deletions, genes in this region do not need to be complemented in thepackaging cell for in vitro models; in in vivo models, the impact uponimmunogenicity of the recombinant virus can be assessed on an ad hocbasis.

The set or library of specific adapter plasmids or pool(s) of adapterplasmids is converted to a set or library of adenoviral vectors. Theadapter plasmids containing the sample nucleic acids are linearized andtransfected into an E1-complementing cell line. The adapter plasmids arepreferably seeded in microtiter tissue culture plates with 96, 384,1,536 or more wells per plate, together with helper plasmids havingsequences homologous to sequences in the adapter plasmid and containingall adenoviral genes necessary for replication and packaging.Recombination occurs between the homologous sequences shared by adapterand helper plasmids to generate an E1-deleted, replication-defectiveadenoviral genome that is replicated and packaged, preferably, in anE1-complementing cell line. If more than one helper plasmid is used,recombination between homologous regions shared among the helperplasmids and recombination between the helper plasmids and adapterplasmid results in the formation of a replication-defective recombinantadenoviral genome. The regions of sequence overlap between the adapterand helper plasmids are at least about a few hundred nucleotides orgreater. Recombination efficiency will increase by increasing the sizeof the overlap.

The E1-functions provided by the trans complementing packaging cellpermit the replication and packaging of the E1-deleted recombinantadenoviral genome. The adapter and/or helper plasmids preferably have nosequence overlap amongst themselves or with the complementing sequencesin the packaging cells that can lead to the formation of RCA.Preferably, at least one of the ITRs on the adapter and helper plasmidsis flanked by a restriction enzyme recognition site not present in theadenoviral sequences or expression cassette so that the ITR is freedfrom vector sequences by digestion of the DNA with that restrictionenzyme. In this way, initiation of replication occurs more efficiently.In order to increase the efficiency of recombinant adenoviralgeneration, higher throughput can be obtained by using microtiter tissueculture plates with 96, 384, or 1,536 wells per plate instead of usinglarge tissue culture vials or flasks. E1-complementing cell lines aregrown in microtiter plates and cotransfected with the libraries ofadapter plasmids and a helper plasmid(s). Automation of the methodusing, for example, robotics can further increase the number ofadenoviral vectors that can be produced (Hawkins et al., (1997) Science276(5320): 1887–9, Houston, (1997) Methods Find. Exp. Clin. Pharmacol.19 Suppl. A:43–5).

As an alternative to the adapter plasmids, the sample nucleic acids canbe ligated to “minimal” adenoviral vector plasmids. The so-called“minimal” adenoviral vectors, according to the present invention, retainat least a portion of the viral genome that is required forencapsidation of the genome into virus particles (the encapsidationsignal). The minimal vectors also retain at least one copy of at least afunctional part or a derivative of the ITR, that is DNA sequencesderived from the termini of the linear adenoviral genome that arerequired for replication. The minimal vectors preferably are used forthe generation and production of helper- and RCA-free stocks ofrecombinant adenoviral vectors and can accommodate up to 38 kb offoreign DNA. The helper functions of the minimal adenoviral vectors arepreferably provided in trans by encapsidation-defective,replication-competent DNA molecules that contain all the viral genesencoding the required gene products, with the exception of those genesthat are present in the complementing cell or genes that reside in thevector genome.

Packaging of the “minimal” adenoviral vector is achieved bycotransfection of an E1-complementing cell line with a helper virus or,alternatively, with a packaging deficient replicating helper system.Preferably, the packaging deficient replicating helper is amplifiedfollowing transfection and expresses the sequences required forreplication and packaging of the minimal adenoviral vectors that are notexpressed by the packaging cell line. The packaging deficientreplicating helper is not packaged into adenoviral particles because itssize exceeds the capacity of the adenoviral virion and/or because itlacks a functional encapsidation signal. The packaging deficientreplicating helper, the minimal adenoviral vector, and the complementingcell line, preferably, have no region of sequence overlap that permitsRCA formation.

The replicating, packaging deficient helper molecule always contains alladenoviral coding sequences that produce proteins necessary forreplication and packaging, with or without the coding sequences providedby the packaging cell line. Replication of the helper molecule itselfmay or may not be mediated by adenoviral proteins and ITRs. Helpermolecules that replicate through the activity of adenoviral proteins(for example, E2-gene products acting together with cellular proteins)contain at least one ITR derived from adenovirus. The E2-gene productscan be expressed by an E1-dependent or an E1-independent promoter. Whereonly one ITR is derived from an adenovirus, the helper molecule alsopreferably contains a sequence that anneals in an intramolecular fashionto form a hairpin-like structure.

Following E2-gene product expression, the adenoviral DNA polymeraserecognizes the ITR on the helper molecule and produces a single-strandedcopy. Then, the 3′-terminus intramolecularly anneals, forming ahairpin-like structure that serves as a primer for reverse strandsynthesis. The product of reverse strand synthesis is a double-strandhairpin with an ITR at one end. This ITR is recognized by adenoviral DNApolymerase that produces a double-stranded molecule with an ITR at bothtermini (see e.g., FIG. 13) and becomes twice as long as the transfectedmolecule (in our example it duplicates from 35 Kb to 70 Kb). Duplicationof the helper DNA enhances the production of sufficient levels ofadenoviral proteins. Preferably, the size of the duplicated moleculeexceeds the packaging capacity of the adenoviral virion and is,therefore, not packaged into adenoviral particles. The absence of afunctional encapsidation signal in the helper molecule further ensuresthat the helper molecule is packaging deficient. The produced adenoviralproteins mediate replication and packaging of the cotransfected orco-infected minimal vectors.

When the replication of the helper molecule is independent of adenoviralE2-proteins, the helper construct preferably contains an origin ofreplication derived from SV40. Transfection of this DNA, together withthe minimal adenoviral vector in an E1-containing packaging cell linethat also inducibly expresses the SV40 Large T protein or as much SV40derived proteins as necessary for efficient replication, leads toreplication of the helper construct and expression of adenoviralproteins. The adenoviral proteins then initiate replication andpackaging of the co-transfected or co-infected minimal adenoviralvectors. Preferably, there are no regions of sequence overlap shared bythe replication-deficient packaging constructs, the minimal adenoviralvectors, and the complementing cell lines that may lead to thegeneration of RCA.

It is to be understood that during propagation of the minimal adenoviralvectors, each amplification step on E1-complementing cells is precededby transfection of any of the described helper molecules since minimalvectors by themselves can not replicate on E1-complementing cells.Alternatively, a cell line that contains all the adenoviral genesnecessary for replication and packaging, which are stably integrated inthe genome and can be excised and replicated conditionally, can be used.(Valerio and Einerhand International patent Appl'n PCT/NL9800061).

Transfection of nucleic acid into cells is required for packaging ofrecombinant vectors into virus particles and can be mediated by avariety of chemicals including liposomes, DEAE-dextran, polybrene, andphosphazenes or phosphazene derivatives (WO97/07226). Liposomes areavailable from a variety of commercial suppliers and include DOTAP®(Boehringer-Mannheim), Tfx®-50, Transfectam®, ProFection® (Promega,Madison, Wis.), and LipofectAmin®, Lipofectin®, LipofectAce® (GibcoBRL,Gaithersburg, Md.). In solution, the lipids form vesicles that associatewith the nucleic acid and facilitate its transfer into cells by fusionof the vesicles with cell membranes or by endocytosis. Othertransfection methods include electroporation, calcium phosphatecoprecipitation, and microinjection. If transfection conditions for agiven cell line have not been established or are unknown, they can bedetermined empirically (Maniatis et al., Molecular Cloning, pages16.30–16.55).

The yield of recombinant adenoviral virus vectors can be increased bydenaturing the double stranded plasmid DNA before transfection into anE1 complementing cell line. Denaturing can occur by heatingdouble-stranded DNA at, for example, 95–100° C., followed by rapidcooling using various ratios of the adapter and helper plasmids thathave overlapping sequences. Also, a PER.C6 derivative that stably ortransiently expresses E2A (DNA binding protein) and/or E2B gene(pTP-Pol) could be used to increase the adenoviral production per wellby increasing the replication rate per cell. Alternatively,cotransfection of recombinase protein(s), recombinase DNA expressionconstruct(s), i.e. recombinase from Kluyveromyces waltii (Ringrose etal., (1997) Eur. J. Biochem. 248(3):903–912), or any other gene or genesencoding factors that can increase homologous recombination efficiencycan be used. The inclusion of 0.1–100 mM sodium butyrate duringtransfection and/or replication of the packaging cells can increaseviral production. These improvements will result in improved viralyields per microtiter well. Therefore, the number and type of assaysthat can be done with one library will increase and may overcomevariability between the various genes or sample nucleic acids in alibrary.

The cell lines used for the production of adenoviral vectors thatexpress E1 region products includes, for example, 293 cells, PER.C6(ECACC 96022940), or 911 cells. Each of these cell lines has beentransfected with nucleic acids that encode for the adenoviral E1 region.These cells stably express E1 region gene products and have been shownto package E1-deleted recombinant adenoviral vectors. Yields ofrecombinant adenovirus obtained on PER.C6 cells are higher than obtainedon 293 cells.

Each of these cell lines provides the basis for introduction of E2B,E2A, or E4 constructs (e.g., ts125E2A and/or hrE2A) that permit thepropagation of adenoviral vectors that have mutations, deletions, orinsertions in the corresponding genes. These cells can be modified toexpress additional adenoviral gene products by the introduction ofrecombinant nucleic acids that stably express the appropriate adenoviralgenes or recombinant nucleic acids and that transiently express theappropriate gene(s), for example, the packaging deficient replicatinghelper molecules or the helper plasmids.

All (or nearly all) trans complementing cells grown in microtiter platewells (96, 384, or more than 1,536 wells) produce recombinant adenovirusfollowing transfection with either the adapter plasmid or the minimaladenoviral plasmid library and the appropriate helper molecule(s). Alarge number of adenoviral gene transfer vectors or a library, eachexpressing a unique gene, can thus be conveniently produced on a scalethat allows analysis of the biological activity of the particular geneproducts both in vitro and in vivo. Due to the wide tissue tropism ofadenoviral vectors, a large number of cell and tissue types aretransducable with an adenoviral library.

Libraries of genes or sample nucleic acids preferably are converted toRCA free adenoviral libraries using the above methods. The adenovirallibraries with unknown function are then used to perform high throughputscreening involving a number of in vitro assays, such as immunologicalassays including ELISAs, proliferation assays, drug resistance assays,enzyme activity assays, organ cultures, differentiation assays, andcytotoxicity assays. Adenoviral libraries can be tested on tissues,tissue sections, or tissue derived primary short-lived cell culturesincluding primary endothelial and smooth muscle cell cultures (Wijnberget al., (1997) Thromb Haemost 78(2), 880–6), coronary artery bypassgraft libraries (Vassalli et al., (1997) Cardiovasc Res. 35(3), 459–69;Fuster and Chesebro, (1985) Adv. Prostaglandin Thromboxane Leukot Res.13, 285–99), umbilical cord tissue including HUVEC (Gimbrone, (1976)Prog. Hemost. Thromb. 3, 1–28; Striker et al., (1980) Methods Cell.Biol. 21A, 135–51), couplet hepatocytes (Graf et al., (1984) Proc. Natl.Acad. Sci. USA 81(20), 6516–20), and epidermal cultures (Fabre, (1991)Immunol. Lett. 29(1–2), 161–5; Phillips, (1991) Transplantation 51(5),937–41). Plant cell cultures, including suspension cultures, can also beused as host cells for the adenoviral libraries carrying any DNAsequence, including human derived DNA sequences and plant derivedsequences. (de Vries et al., (1994) Biochem. Soc. Symp. 60, 43–50;Fukada et al., (1994) Int. J. Devel. Biol. 38(2), 287–99; Jones, (1983)Biochem. Soc. Symp. 48, 221–32; Kieran et al., (1997) J. Biotechnol.59(1–2), 39–52; Stanley, (1993) Curr. Opin. Genet. Dev. 3(1), 91–6;Taticek et al., (1994) Curr. Opin. Biotechnol. 5(2), 165–74.

Depending on the size of the initial unselected library, once anadenoviral library of genes has been reduced to a reasonable number ofcandidates by in vitro assays, the adenoviruses can be tested inappropriate animal models. Examples of animal models that can be usedinclude models for Alzheimer's disease, arteriosclerosis, cancermetastasis, and Parkinson's disease. In addition, transgenic animalswhich have altered expression of endogenous or exogenous genes includingmice with gene(s) that have been inactivated, animals with cancersimplanted at specific sites, human bone marrow chimeric mice such asNOD-SCID mice, and the like can be used. As additional testing isrequired, the stocks of candidate adenoviruses can be increased bypassaging the adenoviruses under the appropriate transcomplementingconditions.

Depending on the animal model used, adenoviral vectors or mixtures ofpre-selected pools of adenoviral vectors can be applied or administeredat appropriate sites such as lung in non-human primates (Sene et al.,(1995) Hum. Gene Ther. 6(12):1587–93) and brain of normal and apoEdeficient mice (Robertson et al., (1998) Neuroscience 25 82(1):171–80.)for Alzheimer's disease (Walker et al., (1997) Brain Res. Brain Res.Rev. 25(1):70–84) and Parkinson disease models (Hockman et al., (1971)Brain Res. 35(2):613–8.; Zigmond and Stricker, (1984) Life Sci.35(1):5–18.). The adenoviral vectors or mixtures of pre-selected poolsof adenoviral vectors can also be injected in the blood stream for liverdisease models including liver failure and Wilson disease (Cuthbert,(1995) J. Investig. Med. 43(4):323–36; Karrer et al., (1984) Curr. Surg.41(6):464–7) and tumor models including metastases models (Esandi etal., (1997) Gene Ther. 4(4):280–7; Vincent et al., (1996) J. Neurosug.85(4):648–54; Vincent et al., (1996) Hum. Gene Ther. 7(2):197–205). Inaddition, selected adenoviral vectors can be injected directly into thebone marrow of human chimeric NOD-SCID mice (Dick et al., (1997) StemCells 15 Suppl. 1: 199–203; Mosier et al., (1988) Nature335(6187):256–9). Finally, selected adenovirus can be applied locally,for example, in vascular tissue of restenosis animal models (Karas etal., (1992) J. Am. Coll. Cardiol. 20(2):467–74).

In addition, in vitro assays can be complemented by using an electronicversion of the sequence database on which the adenoviral library isbuilt. This allows, for example, protein motif searching whereby newmembers of a family can be linked to known members of the same familywith known functions. The use of Hidden Markow Models (HMMs) (Eddy(1996) Proc. Natl. Acad. Sci. USA 94(4):1414–1419) allows theestablishment of novel families by identifying essential features of afamily and building a model of what the members should look like. Thiscan be combined with structural data by using the threading approachwhich uses a known structure as the thread and tries to find a putativestructure without having determined the actual structure of the novelprotein (Rastan and Beeley (1997) Curr. Opin. Genet. Dev. 7 (6):777–83).The functional data, which is obtained using adenoviral libraries madein accordance with the methods disclosed in this application, is thefoundation of the endeavor to find novel genes with expected or desiredfunctions and will be the core of functional genomics. Finally, once thenumber of adenoviral vectors has reached a level at which animalexperiments can be performed, another addition to the method is toproduce the selection of candidate adenoviral vectors carrying thecandidate genes. Then, the clones can be purified by, for example, usingadenovirus tagged in the Hi loop of the knob domain of the fiber.Alternatively, large scale HPLC analysis can be used in asemipreparative fashion to yield partially purified adenoviral samplesfor in vivo or in vitro experiments when more purified adenoviralpreparations are desired. Therefore, the described method and reagentsallow rapid transfer of a collection of genes in in vivo studies of alimited number of animals, which otherwise would be unfeasible. Theautomation of the steps of the procedure using robotics will furtherenhance the number of genes and sample nucleic acids that can befunctionated.

In one aspect, the invention provides a method of producing arecombinant adenoviral vector library. The method includes growing acell culture containing a plurality of cells including adenoviralE1-complementing sequences with i) an adapter plasmid library includingan adapter plasmid based on or derived from an adenovirus having no E1region sequences which overlap with E1 region sequences in the pluralityof cells or a recombinant nucleic acid to be inserted into the packagingcell and would lead to generation of RCA in the plurality of cells, andno E2B region sequences other than essential E2B sequences, no E2Aregion sequences, no E3 region sequences and no E4 region sequences andhaving in operable configuration a functional ITR, a functionalencapsidation signal, and sufficient adenoviral sequences which allowfor homologous recombination with the recombinant nucleic acid, and alibrary of sample nucleic acids inserted into the adapter plasmidoperatively linked to a promoter; and ii) a recombinant nucleic acidbased on or derived from an adenovirus, wherein the recombinant nucleicacid includes in operable configuration a functional ITR and sufficientadenoviral sequences for replication, wherein the recombinant nucleicacid partially overlaps with the adapter plasmid library which allow forhomologous recombination leading to replication-defective, recombinantadenovirus; under conditions whereby a recombinant adenoviral vectorlibrary is produced. Preferably, at least one of the adapter plasmidlibrary and the recombinant nucleic acid are heat denatured prior totransfecting the plurality of cells or ancestors of the plurality ofcells.

Preferably, the adenoviral E1-complementing sequences, the adapterplasmid library and the recombinant nucleic acid have no overlappingsequences which allow for homologous recombination leading toreplication competent virus in a cell into which they are transferred.

In another aspect, the invention provides a method of producing arecombinant adenoviral vector library. The method includes growing acell culture containing a plurality of cells including adenoviral E1complementing sequences with i) a recombinant nucleic acid libraryincluding a first recombinant nucleic acid based on or derived from anadenovirus, including in operable configuration two functional ITRs, onefunctional encapsidation signal, and having no functional adenoviralgenes and a library of sample nucleic acids inserted into the firstrecombinant nucleic acid operatively linked to a promoter; and ii) asecond recombinant nucleic acid based on or derived from an adenovirusincluding in operable configuration two functional ITRs, and sufficientadenoviral sequences for replication, wherein the second recombinantnucleic acid includes a deletion of at least the E1 region andencapsidation signal of the adenovirus; under conditions whereby arecombinant adenoviral vector library is produced. Preferably, the cellculture is in a multiwell format.

Furthermore, the adenoviral E1-complementing sequences, the firstrecombinant nucleic acid and the second recombinant nucleic acidpreferably have no overlapping sequences which allow for homologousrecombination leading to replication competent virus in a cell intowhich they are transferred. Preferably, the cell culture is a PER.C6cell culture.

In one example, growth medium of the cell culture contains sodiumbutyrate in an amount sufficient to enhance production of therecombinant adenoviral vector library.

Preferably, the plurality of cells further includes at least one of anadenoviral preterminal protein and a polymerase complementing sequence.Preferably, the plurality of cells further includes an adenoviral E2complementing sequence. Preferably, the E2 complementing sequence is anE2A complementing sequence or an E2B complementing sequence. In oneaspect, the plurality of cells further includes a recombinase protein,whereby the homologous recombination leading to replication-defective,recombinant adenovirus is enhanced. Preferably, the recombinase proteinis a Kluyveromyces waltii recombinase.

In another aspect, the plurality of cells further includes a nucleotidesequence coding for a recombinase protein. Preferably, the recombinaseprotein is Kluyveromyces waltii recombinase.

In one aspect, the members of the recombinant adenoviral vector libraryare identical.

In one aspect, the promoter is an inducible promoter. Preferably, thepromoter is repressed or down modulated by an adenoviral E1 geneproduct. In one aspect, the promoter includes an AP1 dependent promoter.Preferably, the AP1 dependent promoter is derived from a collagenase, ac-myc, a monocyte chemoattractant protein or a stromelysin gene.

In one aspect, the sample nucleic acids encode a product of unknownfunction. In another aspect, the sample nucleic acids are selected fromthe group consisting of synthetic oligonucleotides, DNAs, cDNAs, genes,ESTs, antisense nucleic acids, or genetic suppressor elements.

In another aspect, the invention provides a method for assigning afunction to products encoded by sample nucleic acids. The methodincludes growing a host cell containing a recombinant adenoviral vectorlibrary produced according to the method of the invention, wherebyproducts encoded by the sample nucleic acids are expressed to produce atleast one altered phenotype in the host cell; and identifying the atleast one altered phenotype, whereby a function is assigned to theproducts encoded by the sample nucleic acids. Preferably, the host cellis a plant cell or an animal cell. Preferably, the animal cell is ahuman cell. In one aspect, the host cell is a member of a cell culture.Preferably, the cell culture is in a multiwell format. Preferably, amethod of the invention is automated.

The invention further provides a non-human host cell containing arecombinant replication-defective adenoviral vector library. Theinvention further provides a non-human host cell containing arecombinant replication-defective adenoviral vector library, wherein thereplication-defective adenoviral vector library is produced by themethod according to a method of the invetion.

The invention further provides an isolated host cell containing areplication-defective adenoviral vector library, such as one wherein thereplication-defective adenoviral vector library is produced by themethod according to the invention. Preferably, the host cell is a humancell.

The invention further provides a method of producing a recombinantadenoviral vector library. This method includes growing a cell culturecontaining a plurality of cells expressing adenoviral E1-regionsequences and expressing one or more functional gene products encoded byat least one adenoviral region selected from an E2A region and an E4region with i) an adapter plasmid library including an adapter plasmidbased on or derived from an adenovirus having no E1 region sequenceswhich overlap with E1 region sequences in the plurality of cells or arecombinant nucleic acid to be inserted into the packaging cell, and noE2B region sequences other than essential E2B sequences, no E2A regionsequences, no E3 region sequences and no E4 region sequences and havingin operable configuration a functional ITR, a functional encapsidationsignal, and sufficient adenoviral sequences which allow for homologousrecombination with the recombinant nucleic acid, and a library of samplenucleic acids inserted into the adapter plasmid operatively linked to apromoter; and ii) a recombinant nucleic acid based on or derived from anadenovirus having no E1 region sequences which overlap with E1 sequencesin the plurality of cells, and having no E2A region sequences or E4region sequences expressed in the plurality of cells which would lead toproduction of RCA and having in operable configuration a functionaladenoviral ITR and sufficient adenoviral sequences for replication inthe plurality of cells, wherein the recombinant nucleic acid hassufficient overlap with the adapter plasmid to provide for homologousrecombination leading to production of recombinant adenovirus in thepackaging cell.; under conditions whereby a recombinant adenoviralvector library is produced in the plurality of cells.

Preferably, the recombinant nucleic acid further has no E3 regionsequences. Preferably, the plurality of cells expresses at least onefunctional E2A gene product. Preferably, the at least one functional E2Agene product is a mutated gene product. Preferably, the mutated geneproduct is temperature sensitive.

Preferably, at least one of the adapter plasmid library and therecombinant nucleic acid are heat denatured prior to transfecting theplurality of cells or ancestors of the plurality of cells.

Preferably, the plurality of cells expresses one or more functional geneproduct encoded by E2B region sequences and wherein E2B region sequencesfor the functional E2B region gene products, other than those requiredfor virus generation, are deleted from the recombinant nucleic acid, andoptionally up to all E2B gene region sequences are deleted from theadapter plasmid.

Preferably, the plurality of cells expresses all gene products encodedby E2B region sequences, and wherein E2B region sequences for thefunctional E2B region gene products, other than those required for virusgeneration, are deleted from the recombinant nucleic acid, andoptionally up to all E2B gene region sequences are deleted from theadapter plasmid. Preferably, the cell culture is a PER.C6 cell culture.Preferably, the promoter is an inducible promoter.

In one aspect, the invention provides a plurality of cells containing arecombinant replication-defective adenoviral vector library, wherein therecombinant replication-defective adenoviral vector library is producedaccording to a method of the invention. Preferably, the plurality ofcells are PER.C6 cells.

The invention further provides a recombinant nucleic acid including: anucleic acid based on or derived from an adenovirus having no E1 regionsequences, which would lead to production of RCA in a packaging cell,into which it is introduced and having in operable configuration afunctional adenoviral ITR and sufficient adenoviral sequences forreplication in the packaging cell, wherein the nucleic acid hassufficient overlap with an adapter plasmid to provide for homologousrecombination leading to production of recombinant adenovirus in thepackaging cell. Preferably, the recombinant nucleic acid has at leastone of no E2A region sequences or no E4 region sequences that areexpressed in the packaging cell and would lead to production ofrecombinant adenovirus in the packaging cell. Preferably, therecombinant nucleic acid has no E2B region sequences, other thanessential E2B region sequences for virus generation, which are expressedin the packaging cell. Preferably, the recombinant nucleic acid has noE3 region sequences. Preferably, the sufficient overlap is about 10 bpto about 5000 bp. Preferably, the sufficient overlap is about 2000 bp toabout 3000 bp. Preferably, the sufficient overlap includes E2B regionsequences essential for virus generation.

The invention further provides an adapter plasmid including a nucleicacid based on or derived from an adenovirus having no E1 regionsequences which overlap with E1 region sequences in a packaging cellinto which it is introduced and would lead to production of RCA and noE2B region sequences other than essential E2B sequences, no E2A regionsequences, no E3 region sequences and no E4 region sequences whichoverlap with other nucleic acid to be inserted into the packaging cellor contained in the packaging cell, and having in operable configurationa functional ITR, a functional encapsidation signal, and sufficientadenoviral sequences which allow for homologous recombination with theother nucleic acid leading to replication-defective, recombinantadenovirus, and a cloning site or a multiple cloning site.

Preferably, the cloning site or the multiple cloning site is operablylinked to a promoter.

Preferably, the promoter is an inducible promoter. Preferably, thepromoter is repressed or down modulated by an adenoviral E1 geneproduct. Preferably, the promoter includes an AP1 dependent promoter.Preferably, the AP1 dependent promoter is derived from a collagenasegene, a c-myc gene, a monocyte chemoattractant protein gene or astromelysin gene.

Preferably, a library of sample nucleic acids is inserted into themultiple cloning site. Preferably, a method of the invention isautomated.

The invention is further explained through use of the followingillustrative Examples.

EXAMPLES Example 1 Generation of Cell Lines Able to Transcomplement E1Defective Recombinant Adenoviral Vectors

911 Cell Line

A cell line that harbors E1 sequences of adenovirus type 5, able totranscomplement E1 deleted recombinant adenovirus, has been generated(Fallaux et al, (1996) Hum. Gene Ther. 7: 215–222). This cell line wasobtained by transfection of human diploid human embryonic retinoblasts(HER) with pAd5XhoIC that contains nt. 80–5788 of Ad5. One of theresulting transformants was designated 911. This cell line has beenshown to be useful in the propagation of E1 defective recombinantadenovirus and has been found to be superior to 293 cells. Unlike 293cells, 911 cells lack a fully transformed phenotype which most likelyresults in better performance as an adenoviral packaging line. In 911cells the plaque assays can be performed faster (4–5 days instead of8–14 days on 293), monolayers of 911 cells survive better under agaroverlay as required for plaque assays, and there is higher amplificationof E1-deleted vectors.

In addition, unlike 293 cells that were transfected with shearedadenoviral DNA, 911 cells were transfected using a defined construct.Transfection efficiencies of 911 cells are comparable to those of 293.

New Packaging Constructs

Source of Adenoviral Sequences

Adenoviral sequences are derived either from pAd5.SalB, containing nt.80–9460 of human adenoviral type 5, (Bernards et al., (1983) Virology127:45–53) or from wild-type Ad5 DNA. pAd5.SalB was digested with SalIand XhoI, the large fragment was religated, and this new clone was namedpAd5.X/S. The pTN construct (constructed by Dr. R. Vogels, IntroGene,NL) was used as a source for the human PGK promoter and the NEO gene.

Human PGK Promoter and NEO^(R) Gene

Transcription of E1A sequences in the new packaging constructs is drivenby the human PGK promoter (Michelson et al, (1983) Proc. Natl. Acad.Sci. USA 80:472–476); Singer-Sam et al, (1984) Gene 32: 409–417) derivedfrom plasmid pTN (gift of R. Vogels), which uses pUC119 (Vieira et al,(1987) pp. 3–11: Methods in Enzymology, Acad. Press Inc.) as a backbone.This plasmid was also used as a source for the NEO gene fused to theHepatitis B Virus (HBV) poly-adenylation signal.

Fusion of PGK Promoter to E1 Genes (FIG. 1)

In order to replace the E1 sequences of Ad5 (ITR, origin of replication,and packaging signal) with heterologous sequences, E1 sequences (nt.459to nt.960) of Ad5 were amplified by PCR using primers Ea1 (SEQ ID NO:27)and Ea2 (SEQ ID NO:28) (see Table I). The resulting PCR product wasdigested with ClaI, ligated into Bluescript (Stratagene), andpredigested with ClaI and EcoRV, resulting in construct pBS.PCRI.

Vector pTN was digested with restriction enzymes EcoRI (partially) andScaI. The DNA fragment containing the PGK promoter sequences was ligatedinto PBS.PCRI digested with ScaI and EcoRI. The resulting construct,PBS.PGK.PCRI, contains the human PGK promoter operatively linked to Ad5E1 sequences from nt.459 to nt.916.

Construction of pIG.E1A.E1B (FIG. 2)

pIG.E1A.E1B.X contains the E1A and E1B coding sequences under thedirection of the PGK promoter. Since Ad5 sequences from nt.459 tont.5788 are present in this construct, pIX protein of the adenovirus isalso encoded by this plasmid. pIG.E1A.E1B.X was made by replacing theScaI-BspEI fragment of pAT-X/S with the corresponding fragment fromPBS.PGK.PCRI (containing the PGK promoter linked to E1A sequences).

Construction of pIG.E1A.NEO (FIG. 3)

In order to introduce the complete E1B promoter and to fuse thispromoter such that the AUG codon of E1B 21 kDa functions exactly as theAUG codon of NEO^(R), the E1B promoter was amplified using primers Ea3(SEQ ID NO:29) and Ep2 (SEQ ID NO:30), where primer Ep2 introduces aNcoI site into the PCR fragment. The resulting PCR fragment, namedPCRII, was digested with HpaI and NcoI and ligated into pAT-X/S, whichwas predigested with HpaI and NcoI. The resulting plasmid was designatedpAT-X/S-PCR2. The NcoI-StuI fragment of pTN, containing the NEO gene andpart of the Hepatitis B Virus (HBV) poly-adenylation signal, was clonedinto pAT-X/S-PCR2, which had been digested with NcoI and NruI. Theresulting construct was designated pAT-PCR2-NEO. The poly-adenylationsignal was completed by replacing the ScaI-SalI fragment of pAT-PCR2.NEOwith the corresponding fragment of pTN, resulting in pAT.PCR2.NEO.p (A).The ScaI-XbaI of pAT.PCR2.NEO.p (A) was replaced with the correspondingfragment of pIG.E1A.E1B-X, containing the PGK promoter linked to E1Agenes. The resulting construct, named pIG.E1A.NEO, contains Ad5 E1sequences (nt.459 to nt.1713) under the control of the human PGKpromoter.

Construction of pIG.E1A.E1B (FIG. 4)

pIG.E1A.E1B contains nt.459 to nt.3510 of Ad5 that encode the E1A andE1B proteins. The E1B sequences are terminated at the splice acceptor atnt.3511. No pIX sequences are present in this construct.

pIG.E1A.E1B was made as follows: The sequences encoding the N-terminalamino acids of E1B 55 kDa were amplified using primers Eb1 (SEQ IDNO:31) and Eb2 (SEQ ID NO:32), which introduces a XhoI site. Theresulting PCR fragment was digested with BglII and cloned into BlII/NruIof pAT-X/S, thereby obtaining pAT-PCR3. The HBV poly (A) sequences ofpIG.E1A.NEO were introduced downstream of the E1B sequences of pAT-PCR3by exchange of the Xba-SalI fragment of pIG.E1A.NEO and the XbaI XhoIfragment of pAT.PCR3.

Construction of pIG.NEO (FIG. 5)

This construct is of use when established cells are transfected withE1A.E1B constructs and NEO selection is required. Because NEO expressionis directed by the E1B promoter, NEO resistant cells are expected toco-express E1A, which also is advantageous for maintaining high levelsof expression of E1A during long-term culture of the cells. pIG.NEO wasgenerated by cloning the HpaI-ScaI fragment of pAT.PCR2.NEO.p(A) orpIG.E1A.NEO, containing the NEO gene under the control of the Ad5 E1Bpromoter, into pBS digested with EcoRV and ScaI.

Testing of Constructs

The integrity of the constructs pIG.E1A.NEO, pIG.E1A.E1B.X, andpIG.E1A.E1B was assessed by restriction enzyme mapping. Furthermore,parts of the constructs that were obtained by PCR analysis wereconfirmed by sequence analysis. No changes in the nucleotide sequencewere found.

The constructs were transfected into primary BRK (Baby Rat Kidney)cells. pIG.E1A.NEO was tested for its ability to immortalize thesecells. pAd5.XhoIC, pIG.E1A.E1B.X, and pIG.E1A.E1B were tested for theirability to fully transform these cells. Kidneys of 6-day old WAG-Rijrats were isolated, homogenized, and trypsinized. Subconfluent dishes(diameter 5 cm) of the BRK cell cultures were transfected with 1 or 5 μgof pIG.NEO, pIG.E1A.NEO, pIG.E1A.E1B, pIG/E1A.E1B.X, pAd5XhiIC, orpIG.E1A.NEO together with PDC26 (Elsen et al, (1983) Virology128:377–390) carrying the Ad5.E1B gene under control of the SV40 earlypromoter. Three weeks post-transfection, when foci were visible, thedishes were fixed, Giemsa stained, and the foci counted.

An overview of the generated adenoviral packaging constructs, and theirability to transform BRK, is presented in FIG. 6. The results indicatethat the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transformBRK cells in a dose-dependent manner. The efficiency of transformationis similar for both constructs and is comparable to what was found withthe construct, pAd5.XhoIC, that was used to make 911 cells.

As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However,co-transfection of an E1B expression construct (PDC26) resulted in asignificant increase of the number of transformants (18 versus 1),indicating that the E1A encoded by pIG.E1A.NEO is functional. Therefore,it was concluded that the newly generated packaging constructs aresuitable for the generation of new adenoviral packaging lines.

Generation of Cell Lines with New Packaging Constructs Cell Lines andCell Culture

Human A549 bronchial carcinoma cells (Shapiro et al, (1978) Biochem.Biophys. Acta 530:197–207), human embryonic retinoblasts (HER),Ad5-E1-transformed human embryonic kidney (HEK) 293 cells (Graham et al,(1977) J. Gen. Virol. 36: 59–72), Ad5-transformed HER 911 cells (Fallauxet al, (1996). Hum. Gene Ther. 7: 215–222), and PER cells were grown inDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal CalfSerum (FCS) and antibiotics in a 5% CO₂ atmosphere at 37° C. Cellculture media, reagents, and sera were purchased from Gibco Laboratories(Grand Island, N.Y.). Culture plastics were purchased from Greiner(N,rtingen, Germany) and Corning (Corning, N.Y.).

Viruses and Virus Techniques

The construction of recombinant adenoviral vectors IG.Ad.MLP.nls.lacZ,IG.Ad.MLP.luc, IG.Ad.MLP.TK, and IG.Ad.CMV.TK is described in detail inpatent application EP 95202213. The recombinant adenoviral vectorIG.Ad.MLP.nls.lacZ contains the E. coli lazZ gene, encodingβ-galactosidase, under control of the Ad2 major late promoter (MLP).IG.Ad.MLP.luc contains the firefly luciferase gene driven by the Ad2 MLPand adenoviral vectors. IG.Ad.MLP.TK and IG.Ad.CMV.TK contain the HerpesSimplex Virus thymidine kinase (TK) gene under the control of the Ad2MLP and the Cytomegalovirus (CMV) enhancer/promoter, respectively.

Transfections

All transfections were performed by calcium phosphate DNA precipitation(Graham et al., (1973) Virology 52: 456–467) with the GIBCO CalciumPhosphate Transfection System (GIBCO BRL Life Technologies, Inc.,Gaithersburg, USA), according to the manufacturer's protocol.

Western Blotting

Subconfluent cultures of exponentially growing 293, 911, andAd5-E1-transformed A549 and PER cells were washed with PBS and scrapedin Fos-RIPA buffer (10 mM Tris (pH 7,5), 150 mM NaCl, 1% NP4, 0.01%sodium dodecyl sulfate (SDS), 1% NA-DOC, 0.5 mM phenyl methyl sulfonylfluoride (PMSF), 0.5 mM trypsin inhibitor, 50 mM NaF and 1 mM sodiumvanadate). After 10 min. at room temperature, lysates were cleared bycentrifugation. Protein concentrations were measured with the BioRadprotein assay kit and 25 μg total cellular protein was loaded on a 12.5%SDS-PAA gel. After electrophoresis, proteins were transferred tonitrocellulose (1 h at 300 mA). Prestained standards (Sigma, USA) wererun in parallel. Filters were blocked with 1% bovine serum albumin (BSA)in TBST (10 mM Tris, pH 8, 15 mM NaCl, and 0.05% Tween-20) for 1 hour.First antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDa antibodyA1C6 (Zantema et al, unpublished) and the rat monoclonalanti-Ad5-E1B-221-kDa antibody C1G11 (Zantema et al, (1985) Virology142:44–58). The second antibody was a horseradish peroxidase-labelledgoat anti-mouse antibody (Promega). Signals were visualized by enhancedchemoluminescence (Amersham Corp. UK).

Southern Blot Analysis

High molecular weight DNA was isolated and then 10 μg of the DNA wasdigested to completion and fractionated on a 0.7% agarose gel. Southernblot transfer to Hybond N⁺ (Amersham, UK) was performed with a 0.4 MNaOH, 0.6 M NaCl transfer solution (Church and Gilbert, 1984).Hybridization was performed with a 2463-nt SspI-HindIII fragment frompAd5.SalB (Bernards et al, (1983) Virology 127:45–53). This fragmentconsisted of Ad5 bp. 342–2805. The fragment was radiolabeled withα-^(32p)=dCTP with the use of random hexanucleotide primers and KelnowDNA polymerase. The southern blots were exposed to a Kodak XAR-5 film at−80° C. and to a Phospho-Imager screen, which was analyzed by B&Lsystems Molecular Dynamics Software.

A549

Ad5-E1-transformed A549 human bronchial carcinoma cell lines weregenerated by transfection with pIG.E1A.NEO and selection for G418resistance. Thirty-one G418 resistant clones were established.Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418 resistantcell lines.

PER

Ad5-E1-transformed human embryonic retina (HER) cells were generated bytransfection of primary HER cells with plasmid pIG.E1A.E1B. Transformedcell lines were established from well-separated foci. Seven clonal celllines, PER.Cl, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8, and PER.C9, wereestablished. One of the PER clones, namely PER.C6, has been deposited atthe ECACC under number 96022940.

Expression of Ad5 E1A and E1B Genes in Transformed A549 and PER Cells

Expression of the Ad5 E1A and the 55 kDa and 21 kDa E1B proteins in theestablished A549 and PER cells was analyzed by Western blotting, withthe use of monoclonal antibodies (mAb). mAb M73 recognizes the E1Aproducts, whereas mAbs AIC6 and C1G11 are directed against the 55 kDaand 21 kDa E1B proteins, respectively. The antibodies did not recognizeproteins in extracts from the parental A549 or the primary HER cells(data not shown). None of the A549 clones that were generated byco-transfection of pIG.NEO and pIG.E1A.E1B expressed detectable levelsof E1A or E1B proteins (not shown). Some of the A549 clones that weregenerated by transfection with pIG.E1A.NEO expressed the Ad5 E1Aproteins (FIG. 7), but the levels were much lower than those detected inprotein lysates from 293 cells. The steady state E1A levels detected inprotein extracts from PER cells were much higher than those detected inextracts from A549-derived cells. All PER cell lines expressed similarlevels of E1A proteins (FIG. 7). The expression of the E1B proteins,particularly in the case of E1B 55 kDa, was more variable. Compared to911 and 293, the majority of the PER clones express high levels of E1B55 kDa and 2 kDa. The steady state level of E1B 21 kDa was the highestin PER.C3. None of the PER clones lost expression of the Ad5 E1 genesupon serial passage of the cells (not shown). The level of E1 expressionin PER cells was found to remain stable for at least 100 populationdoublings. The PER clones were characterized in more detail.

Southern Analysis of PER Clones

To study the arrangement of the Ad5-E1 encoding sequences in the PERclones Southern analyses were performed. Cellular DNA was extracted fromall PER clones, and from 293 and 911 cells. The DNA was digested withHindIII, which cuts once in the Ad5 E1 region. Southern hybridization onHindIII-digested DNA, using a radiolabeled Ad5-E1-specific probe,revealed the presence of several integrated copies of pIG.E1A.E1B in thegenome of the PER clones. FIG. 8 shows the distribution pattern of E1sequences in the high molecular weight DNA of the different PER celllines. The copies were concentrated in a single band, which suggeststhat they were integrated as tandem repeats. In the case of PER.C3, C5,C6 and C9, additional hybridizing bands of low molecular weight werefound that indicate the presence of truncated copies of pIG.E1A.E1B. Thenumber of copies was determined with the use of a Phospho-Imager.PER.C1, C3, C4, C5, C6, C8 and C9 were estimated to contain 2, 88, 5, 4,5, 5, and 3 copies of the Ad5 E1 coding region, respectively, and 911and 293 cells contain 1 and 4 copies of the Ad5 E1 sequences,respectively.

Transfection Efficiency

Recombinant adenovectors are generated by co-transfection of adaptorplasmids and the large ClaI fragment of Ad5 into 293 cells (EPO patentapplication 95202213). The recombinant virus DNA is formed by homologousrecombination between the homologous viral sequences that are present inthe plasmid and the adenoviral DNA. The efficacy of this method, as wellas that of alternative strategies, is highly dependent on thetransfectability of the helper cells. Therefore, the transfectionefficiencies of some of the PER clones were compared with 911 cells,using the E. coli β-galactosidase-encoding lacZ gene as a reporter (FIG.9).

Production of Recombinant Adenovirus

Yields of recombinant adenovirus obtained after inoculation of 293, 911,PER.C3, PER.C5, and PER.C6 with different adenoviral vectors arepresented in Table II.

The results indicate that the recombinant adenoviral vector yieldsobtained with PER cells are at least as high as those obtained with theexisting cell lines. In addition, the yields of the novel adenoviralvector IG.Ad.MLPI.TK are similar or higher than the yields obtained forthe other viral vectors on all cell lines tested.

Generation of New Adenoviral Vectors (FIG. 10)

The recombinant adenoviral vectors used (see EPO patent application EP95202213) are deleted for E1 sequences from 459 to nt. 3328. Asconstruct pE1A.E1B contains Ad5 sequences 459 to nt. 3510, there is asequence overlap of 183 nt. between E1B sequences in the packagingconstruct pIG.E1A.E1B and recombinant adenoviruses, for example,IG.Ad.MLP.TK. The overlapping sequences were deleted from the newadenoviral vectors. In addition, non-coding sequences derived from lacZ,which are present in the original constructs, were deleted as well. Thiswas achieved (see FIG. 10) by PCR amplification of the SV40 poly (A)sequences from pMLP.TK using primers SV40-1 (SEQ ID NO: 33) and SV40-2(SEQ ID NO: 34). In addition, Ad5 sequences present in this constructwere amplified from nt. 2496 (Ad5, introduces a BglII site) to nt. 2779(Ad5-2). Both PCR fragments were digested with BglII and were ligated.The ligation product was PCR amplified using primers SV40-1 and Ad5-2(SEQ ID NO:36). The PCR product obtained was cut with BamHI and AflIIand was ligated into pMLP.TK, which was predigested with the sameenzymes. The resulting construct, named pMLPI.TK, contains a deletion inadenoviral E1 sequences from nt. 459 to nt. 3510.

Packaging System

The combination of the new packaging construct pIG.E1A.E1B and therecombinant adenovirus pMLPI.TK, which do not have any sequence overlap,are presented in FIG. 11. In this FIG., the original situation is alsopresented, where the sequence overlap is indicated. The absence ofoverlapping sequences between pIG.E1A.E1B and pMLPI.TK (FIG. 11 a)excludes the possibility of homologous recombination between thepackaging construct and the recombinant virus, and is therefore asignificant improvement for production of recombinant adenovirus.

In FIG. 11 b the situation is depicted for pIG.E1A.NEO andIG.Ad.MLPI.TK. pIG.E1A.NEO when transfected into established cells, isexpected to be sufficient to support propagation of E1-deletedrecombinant adenovirus. This combination does not have any sequenceoverlap, preventing generation of RCA by homologous recombination. Inaddition, this convenient packaging system allows the propagation ofrecombinant adenoviruses that are deleted just for E1A sequences and notfor E1B sequences.

Recombinant adenoviruses expressing E1B in the absence of E1A areattractive because the E1B protein, in particular E1B 19 kDa, is able toprevent infected human cells from lysis by tumor necrosis factor (TNF).Gooding et al, (1991) J. Virol. 65: 3083–3094).

Generation of Recombinant Adenovirus Derived from pMLPI.TK

Recombinant adenovirus was generated by co-transfection of 293 cellswith SalI linearized pMLPI.TK DNA and ClaI linearized Ad5 wt DNA. Theprocedure is schematically represented in FIG. 12.

Example 2 Plasmid-based System for Rapid RCA-free Generation ofRecombinant Adenoviral Vectors

Construction of Adenoviral Clones pBr/Ad.Bam-rITR (ECACC DepositP97082122)

In order to facilitate blunt end cloning of the ITR sequences, wildtypehuman adenovirus type 5 (Ad5) DNA was treated with Klenow enzyme in thepresence of excess dNTPs. After inactivation of the Klenow enzyme,purification by phenol/chloroform extraction, and ethanol precipitation,the DNA was digested with BamHI. This DNA preparation was used withoutfurther purification in a ligation reaction with pBr322 derived vectorDNA. The pBr322 DNA was prepared as follows: pBr322 DNA was digestedwith EcoRV and BamHI, dephosphorylated by treatment with TSAP enzyme(Life Technologies), and purified on LMP agarose gel (SeaPlaque GTG).After transformation into competent E.coli DH5a (Life Techn.) andanalysis of ampicillin resistant colonies, one clone was selected thatshowed a digestion pattern consistent with an insert extending from theBamHI site in Ad5 to the right ITR. Sequence analysis of the cloningborder at the right ITR revealed that the G residue closest to the 3′end of the ITR was missing. However, the remainder of the ITR was foundto be correct. The missing G residue is complemented by the other ITRduring replication.

pBr/Ad.Sal-rITR (ECACC Deposit P97082119)

pBr/Ad.Bam-rITR was digested with BamHI and SalI. The vector fragmentincluding the adenoviral insert was isolated in LMP agarose (SeaPlaqueGTG), ligated to a 4.8 kb SalI-BamHI fragment obtained from wt Ad5 DNA,and purified with the Geneclean II kit (Bio 101, Inc.). One clone waschosen and the integrity of the Ad5 sequences was determined byrestriction enzyme analysis. Clone pBr/Ad.Sal-rITR contains adeno type 5sequences from the SalI site at bp 16746 up to and including the rITR(missing the G residue closest to the 3′ end).

pBr/Ad.Cla-Bam (ECACC Deposit P97082117)

Wildtype adeno type 5 DNA was digested with ClaI and BamHI, and the 20.6kb fragment was isolated from gel by electro-elution. pBr322 wasdigested with the same enzymes and purified by agarose gel fromGeneclean. Both fragments were ligated and transformed into competentDH5α. The resulting clone pBr/Ad.Cla-Bam was analyzed by restrictionenzyme digestion and shown to contain an insert with adenoviralsequences from bp 919 to 21566.

pBr/Ad.AflII-Bam (ECACC Deposit P97082114)

Clone pBr/Ad.Cla-Bam was linearized with EcoRI (in pBr322) and partiallydigested with AflII. After heat inactivation of AflII for 20 minutes at65° C., the fragment ends were filled in with Klenow enzyme. The DNA wasthen ligated to a blunt double stranded oligo linker containing a PacIsite (5′-AATTGTCTTAATTAACCGCTTAA-3′) (SEQ ID NO:1). This linker was madeby annealing the following two oligonucleotides:5′-AATTGTCTTAATTAACCGC-3′ (SEQ ID NO:2) and 5′-AATTGCGGTTAATTAAGAC-3′(SEQ ID NO:3), followed by blunting with Klenow enzyme. Afterprecipitation of the ligated DNA to change buffer, the ligations weredigested with an excess PacI enzyme to remove concatameres of the oligo.The 22016 bp partial fragment, containing Ad5 sequences from bp 3534 upto 21566 and the vector sequences, was isolated in LMP agarose(SeaPlaque GTG), religated, and transformed into competent DH5α. Oneclone that was found to contain the PacI site and that had retained thelarge adeno fragment was selected and sequenced at the 5′ end to verifycorrect insertion of the PacI linker in the (lost) AflII site.

pBr/Ad.Bam-rITRpac#2 (ECACC Deposit P97082120) and pBr/Ad.Bam-rITR#8(ECACC Deposit P97082121)

To allow insertion of a PacI site near the ITR of Ad5 in clonepBr/Ad.Bam-rITR, about 190 nucleotides were removed between the ClaIsite in the pBr322 backbone and the start of the ITR sequences.pBr/Ad.Bam-rITR was digested with ClaI and treated with nuclease Bal31for varying lengths of time (2, 5, 10, and 15 minutes). The extent ofnucleotide removal was followed by separate reactions on pBr322 DNA(also digested at the ClaI site), using identical buffers andconditions. Bal31 enzyme was inactivated by incubation at 75° C. for 10minutes, the DNA was precipitated, and then the DNA was resuspended in asmaller volume of TE buffer. To ensure blunt ends, the DNA was furthertreated with T4 DNA polymerase in the presence of excess dNTPs. Afterdigestion of the (control) pBr322 DNA with SalI, satisfactorydegradation (˜150 bp) was observed in the samples treated for 10 or 15minutes. The 10 or 15 minute treated pBr/Ad.Bam-rITR samples were thenligated to the above-described blunted PacI linkers (seepBr/Ad.AflII-Bam). Ligations were purified by precipitation, digestedwith excess PacI, and separated from the linkers on an LMP agarose gel.After religation, DNAs were transformed into competent DH5α and coloniesanalyzed. Ten clones that showed a deletion of approximately the desiredlength were selected and were further analyzed by T-track sequencing (T7sequencing kit, Pharmacia Biotech). Two clones were found with the PacIlinker inserted just downstream of the rITR. After digestion with PacI,clone #2 had 28 bp and clone #8 had 27 bp attached to the ITR.

pWE/Ad.AflII-rITR (ECACC Deposit P97082116)

Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts.First, a linker containing a unique PacI site was inserted in the EcoRIsites of pWE15, creating pWE.pac. The double stranded PacI oligo wasused as described for pBr/Ad.AflII-BamHI, except that its EcoRIprotuding ends were used. The following fragments were then isolated byelectro-elution from an agarose gel: pWE.pac digested with PacI,pBr/AflII-Bam digested with PacI and BamHI, and pBr/Ad.Bam-rITR#2digested with BamHI and PacI. These fragments were ligated together andpackaged using λ phage packaging extracts (Stratagene) according to themanufacturer's protocol. After infection into host bacteria, colonieswere grown on plates and analyzed for presence of the complete insert.pWE/Ad.AflII-rITR contains all adenovirus type 5 sequences from bp 3534(AflII site) up to and including the right ITR (missing the G residueclosest to the 3′ end).

Adeno 5 wt DNA was treated with Klenow enzyme in the presence of excessdNTPs and subsequently digested with SalI. Two of the resultingfragments, designated left ITR-Sal(9.4) and Sal(16.7)-right ITR, wereisolated in LMP agarose (Seaplaque GTG). pBr322 DNA was digested withEcoRV and SalI and treated with phosphatase (Life Technologies). Thevector fragment was isolated using the Geneclean method (BIO 101, Inc.)and ligated to the Ad5 SalI fragments. Only the ligation with the 9.4 kbfragment gave colonies with an insert. After analysis and sequencing ofthe cloning border, a clone was chosen that contained the full ITRsequence and extended to the SalI site at bp 9462.

pBr/Ad.1ITR-Sal(16.7) (ECACC Deposit P97082118)

pBr/Ad.1ITR-Sal(9.4) is digested with SalI and dephosphorylated (TSAP,Life Technologies). To extend this clone up to the third SalI site inAd5, pBr/Ad.Cla-Bam was linearized with BamHI and partially digestedwith SalI. A 7.3 kb SalI fragment containing adenoviral sequences from9462–16746 was isolated in LMP agarose gel and ligated to theSalI-digested pBr/Ad.1ITR-Sal(9.4) vector fragment.

pWE/Ad.AflII-EcoRI

pWE.pac was digested with ClaI and the 5′ protruding ends were filled inusing Klenow enzyme. The DNA was then digested with PacI and isolatedfrom an agarose gel. pWE/AflII-rITR was digested with EcoRI and aftertreatment with Klenow enzyme, was digested with PacI. The large 24 kbfragment containing the adenoviral sequences was isolated from anagarose gel and ligated to the ClaI-digested and blunted pWE.pac vectorusing the Ligation Express™ kit from Clontech. After transformation ofUltracompetent XL10-Gold cells from Stratagene, clones were identifiedthat contained the expected insert. pWE/AflII-EcoRI contains Ad5sequences from bp 3534–27336.

Construction of New Adapter Plasmids

The absence of sequence overlap between the recombinant adenovirus andE1 sequences in the packaging cell line is essential for safe, RCA-freegeneration and propagation of new recombinant viruses. The adapterplasmid pMLPI.TK (FIG. 10) is an example of an adapter plasmid designedfor use according to the invention in combination with the improvedpackaging cell lines of the invention. This plasmid was used as thestarting material to make a new vector in which nucleic acid moleculesincluding specific promoter and gene sequences can be easily exchanged.

First, a PCR fragment was generated from pZipΔMo+PyF101(N⁻) template DNA(described in PCT/NL96/00195) with the following primers: LTR-1: 5′-CTGTAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3′ (SEQ IDNO:4) and LTR-2: 5′-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGCGTT AAC CGG GCG ACT CAG TCA ATC G-3′ (SEQ ID NO:5). Pwo DNA polymerase(Boehringer Mannheim) was used according to the manufacturer's protocolwith the following temperature cycles: 1 cycle of 5 minutes at 95° C., 3minutes at 55° C., and 1 minute at 72° C.; followed by 30 cycles of 1minute at 95° C., 1 minute at 60° C., and 1 minute at 72° C.; followedby 1 cycle of 10 minutes at 72° C. The PCR product was then digestedwith BamHI and ligated into a pMLP10 vector (Levrero et al, (1991) Gene101:195–202) digested with PvuII and BamHI, thereby generating vectorpLTR10. This vector contains adenoviral sequences from bp 1 up to bp454, followed by a promoter that includes part of the Mo-MuLV LTR inwhich the wildtype enhancer sequences are replaced by the enhancer froma mutant polyoma virus (PyF101). The promoter fragment was designatedL420.

Next, the coding region of the murine HSA gene was inserted. pLTR10 wasdigested with BstBI followed by Klenow treatment and digestion withNcoI. The HSA gene was obtained by PCR amplification on pUC18-HSA (Kayet al, (1990) J. Immunol. 145:1952–1959) using the following primers:HSA1, 5′-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3′ (SEQ ID NO:6) andHSA2, 5′-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAGAA-3′(SEQ ID NO:7). The 269 bp amplified fragment was subcloned in ashuttle vector using the NcoI and BglII sites. Sequencing confirmedincorporation of the correct coding sequence of the HSA gene, but withan extra TAG insertion directly following the TAG stop codon. The codingregion of the HSA gene, including the TAG duplication, was then excisedas a NcoI(sticky)-SaII(blunt) fragment and cloned into the 3.5 kbNcoI(sticky)/BstBI(blunt) fragment from pLTR10, resulting in pLTR-HSA10.

Finally, pLTR-HSA10 was digested with EcoRI and BamHI after which thefragment containing the left ITR, packaging signal, L420 promoter, andHSA gene was inserted into vector pMLPI.TK, which was digested with thesame enzymes, thereby replacing the promoter and the gene sequences.This resulted in the new adapter plasmid pAd/L420-HSA (FIG. 19) thatcontains convenient recognition sites for various restriction enzymesaround the promoter and gene sequences. SnaBI and AvrII can be combinedwith HpaI, NheI, KpnI, HindIII to exchange promoter sequences, while thelatter sites can be combined with the ClaI or BamHI sites 3′ from theHSA coding region to replace genes in this construct.

Another adapter plasmid that was designed to allow easy exchange ofnucleic acid molecules was made by replacing the promoter, gene, andpoly A sequences in pAd/L420-HSA with the CMV promoter, a multiplecloning site, an intron, and a poly-A signal. For this purpose,pAd/L420-HSA was digested with AvrII and BglII, followed by treatmentwith Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vectorand adenoviral sequences was isolated and ligated to a blunt 1570 bpfragment from pcDNA1/amp (Invitrogen), which was obtained by digestionwith HhaI and AvrII followed by treatment with T4 DNA polymerase. Thisadapter plasmid was named pCLIP (FIG. 20).

Generation of Recombinant Adenoviruses

E1-deleted Recombinant Adenoviruses with wt E3 Sequences

To generate E1 deleted recombinant adenoviruses with the newplasmid-based system, the following constructs were prepared: an adapterconstruct containing the expression cassette with the gene of interestlinearized with a restriction enzyme that cuts at the 3′ side of theoverlapping adenoviral genome fragment, preferably not containing anypBr322 vector sequences; and a complementing adenoviral genome constructpWE/Ad.AflII-rITR digested with PacI.

These two DNA molecules were further purified by phenol/chloroformextraction and ethanol precipitation. Co-transfection of these plasmidsinto an adenoviral packaging cell line, preferably a cell line accordingto the invention, generates recombinant replication deficientadenoviruses by a one-step homologous recombination between the adapterand the complementing construct (FIG. 21). Alternatively, instead ofpWE/Ad.AflII-rITR, other fragments can be used, e.g., pBr/Ad.Cla-Bamdigested with EcoRI and BamHI or pBr/Ad.AflII-BamHI digested with PacIand BamHI can be combined with pBr/Ad.Sal-rITR digested with SalI. Inthis case, three plasmids are combined and two homologous recombinationsare needed to obtain a recombinant adenovirus (FIG. 22). It is to beunderstood that those skilled in the art may use other combinations ofadapter and complementing plasmids without departing from the presentinvention.

A general protocol as outlined below and meant as a non-limiting exampleof the present invention has been performed to produce severalrecombinant adenoviruses using various adapter plasmids and theAd.AflII-rITR fragment. Adenoviral packaging cells (PER.C6) were seededin ˜25 cm² flasks and were transfected with a mixture of DNA andlipofectamine agent (Life Techn.) as described by the manufacturer whenthey were at about 80% confluency. Routinely, 40 μl lipofectamine, 4 μgadapter plasmid, and 4 μg of the complementing adenoviral genomefragment AflII-rITR (or 2 μg of all three plasmids for the doublehomologous recombination) were used. Under these conditions, transienttransfection efficiencies of approximately 50% (48 hrs posttransfection) were obtained as determined with control transfectionsusing a pAd/CMV-LacZ adapter. Two days later, cells were passaged toabout 80 cm² flasks and further cultured.

Approximately five (for the single homologous recombination) to elevendays (for the double homologous recombination) later, a cytopathiceffect (CPE) was seen, indicating that functional adenovirus had formed.Cells and medium were harvested upon full CPE and recombinant virus wasreleased by freeze/thawing. An extra amplification step in a 80 cm²flask was routinely performed to increase the yield since, at theinitial stage, the titers was found to be variable despite theoccurrence of full CPE. After amplification, viruses were harvested andplaque purified on PER.C6 cells. Individual plaques were tested forviruses with active transgenes.

Four different recombinant adenoviruses, containing the humaninterleukin-3 gene (see FIG. 1, WO88/04691), the human endothelialnitric oxide gene (Janssens et al, (1992) J. Biol. Chem.267:14519–14522), the Tc1A transposase gene (Vos et al, (1993) GenesDev. 7:1244–1253), or the bacterial LacZ gene (Kalderon et al, (1984)Cell 39:499–509, have been produced using this protocol. In all cases,functional adenovirus was formed and all isolated plaques containedviruses with an active transgene.

E1-deleted Recombinant Adenoviruses with Modifications in the E3 or E4Regions

Besides replacements in the E1 region, it is possible to delete the E3region or replace part of the E3 region in the adenovirus because E3functions are not necessary for the replication, packaging, andinfection of a recombinant virus. This creates the opportunity to use alarger insert or to insert more than one gene without exceeding themaximum packagable size (approximately 105% of wt genome length). Thiscan be done, for example, by deleting part of the E3 region in thepBr/Ad.Bam-rITR clone by digestion with XbaI and religation. Thisremoves Ad5 wt sequences 28592–30470 including all known E3 codingregions. Another example is the precise replacement of the coding regionof gp19K in the E3 region with a polylinker allowing insertion of newsequences. The replacement leaves all other coding regions intact,obviates the need for a heterologous promoter because the transgene isdriven by the E3 promoter and pA sequences which leaves more space forcoding sequences, and results in very high transgene expression, atleast as good as in a control E1 replacement vector.

For this purpose, the 2.7 kb EcoRI fragment from wt Ad5 containing the5′ part of the E3 region was cloned into the EcoRI site of pBluescript(KS⁻) (Stratagene). Next, the HindIII site in the polylinker was removedby digestion with EcoRV and HincII and subsequent religation. Theresulting clone, pBS.Eco-Eco/ad5ΔHIII, was used to delete the gp19Kcoding region. Primers 1 (5′-GGG TAT TAG GCC AAAGGCGCA-3′) (SEQ ID NO:8)and 2 (5′-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3′) (SEQ ID NO:9)were used to amplify a sequence from pBS.Eco-Eco/ad5ΔHIII correspondingto sequences 28511 to 28734 in wt Ad5 DNA. Primers 3 (5′-GAT CCC ATG GGGATC CTT TAC TAA GTT ACA AAG CTA-3′) (SEQ ID NO:10) and 4 (5′-GTC GCT GTAGTT GGA CTG G-3′) (SEQ ID NO:11) were used on the same DNA to amplifyAd5 sequences from 29217 to 29476. The two resulting PCR fragments wereligated together by virtue of the newly introduced NcoI site andsubsequently digested with XbaI and MunI. This fragment was then ligatedinto a pBS.Eco-Eco/ad5ΔHIII vector that had been partially digested withXbaI and MunI, generating pBS.Eco-Eco/ad5 ΔHIII.Δgp19K.

To allow insertion of foreign genes into the HindIII and BamHI site, anXbaI deletion was made in pBS.Eco-Eco/ad5ΔHIII.Δgp19K to remove theBamHI sites in the Bluescript polylinker. The resulting plasmidpBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI, contained unique HindIII and BamHIsites corresponding to sequences 28733 (HindIII) and 29218 (BamHI) inAd5. After introduction of a foreign gene into these sites, either thedeleted XbaI fragment is reintroduced or the insert is recloned intopBS.Eco-Eco/ad5 ΔHIII.Δgp19K using HindIII and, for example, MunI. Usingthis procedure, plasmids expressing HSV-TK (McKnight (1980) Nucl. Acid.Res. 8:5949–5964 and Vincent et al (1996) Hum. Gene Ther. 7:197–205),hIL-1α (Esandi et al, (1998) Gene Therapy 5), rat IL-3β (Esandi et al,(1998) Gene 11242), luciferase (DeWit et al, (1987) Mol. Cell Biol.7:725–737), or LacZ were generated. The unique SrfI and NotI sites inthe pBS.Eco-Eco/ad5ΔHIII.Dgp19K plasmid (with or without an insertedgene of interest) are used to transfer the region containing the gene ofinterest into the corresponding region of pBr/Ad.Bam-rITR, yieldingconstruct pBr/Ad.Bam-rITRDgp19K (with or without an inserted gene ofinterest). This construct is used as described supra to producerecombinant adenoviruses. In the viral context, expression of insertedgenes is driven by the adenoviral E3 promoter.

Recombinant viruses that are both E1 and E3 deleted are generated by adouble homologous recombination procedure as described above forE1-replacement vectors using a plasmid-based system which includes: anadapter plasmid for E1 replacement with or without insertion of a firstgene of interest according to the invention, the pWE/Ad.AflII-EcoRIfragment, and the pBr/Ad.Bam-rITRΔgp19K plasmid with or withoutinsertion of a second gene of interest.

In a non-limiting example, the generation and functionality of arecombinant adenovirus containing the murine HSA gene in the E1 regionand the firefly luciferase gene in the gp19K region are described. Theluciferase gene was excised from pAd/MLP-Luc (described in EPO patentappl'n 0707071) as a HindIII-BamHI construct and cloned into theHindIII-BamHI sites of pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI. Then, theMscI-MunI fragment containing the luciferse gene was cloned into thecorresponding sites of pBS.Eco-Eco/ad5Δgp19K, generatingpBS.Eco-Eco/ad5Δgp19K.luc. This restores the Eco-Eco fragment, but nowwith the luciferase gene in place of gp19K.

To simplify further manipulation, the internal EcoRI sites in theluciferase insert were mutated without making changes to the amino acidsequence of the luciferase gene. One EcoRI site flanked the HindIII sitein the 5′ non-coding region of the luciferase insert and the other sitewas located 588 bp 3′ from the starting ATG. A 695 bp PCR product wasgenerated with the following primers: 5′-CGA TAA GCT TAA TTC CTT TGT GTTT-3′ (SEQ ID NO:12) and 5′-CTT AGG TAA CCC AGT AGA TCC AGA GGA GTTCAT-3′ (SEQ ID NO:13) and digested with HindIII and BstEII. Thisfragment was then ligated to HindIII-BstEII, withpBS.Eco-Eco/ad5Δgp19K.luc replacing the corresponding insert in thisvector. The resulting construct is named pBS.Eco-Eco/ad5Δgp19K.luc². Theluciferase gene and part of the E3 region were then excised from thisclone with SrfI and NotI and introduced in the corresponding sites inpBr/Ad.Bam-rITR, generating clone pBr/Ad.Bam-rITRΔgp19K/luc².

The adapter plasmid pAd5/S1800HSA used for the replacement of E1 in thedouble insert virus contains the murine HSA gene driven by a retrovirusLTR-based promoter. This adapter plasmid was generated from thepAd5/L420-HSA construct described infra by replacement of the promotersequence. First, a PCR product was generated on a retroviral vectorbased on the MFG-S vector described in WO 95/34669 using the sameprimers as for the amplification of the L420 promoter fragment(described infra). This amplifies the sequences corresponding to bp453–877 in the MFG-S vector. The L420 promoter in pAd5/IL420-HSA (FIG.21) was then exchanged for the PCR fragment using the unique AvrII andHindlII sites. The resulting construct, pAd5/S430-HSA, was then digestedwith NheI and ScaI and the 4504 bp fragment containing the HSA gene, pAsequences, Ad5 sequences, and vector sequences to the ScaI site in theampicillin gene was isolated.

The construct pAd5/S430-HSA also was digested with XbaI and ScaI and the1252 bp fragment (containing the remainder of the ampicillin gene, theleft ITR and packaging signal from the adenovirus, and the 5′ part ofthe S430 promoter) was isolated. A third fragment of 1576 bp wasisolated from the MFG-S-based retroviral vector following a XbaIdigestion and contained MFG-S sequences corresponding to bp 695–2271.

The adapter plasmid pAd5/S1800-HSA was constructed by ligating the threeisolated fragments. The double insert virus Ad5/S1800-HSA.E31luc wasgenerated (as described above) by transfection of the following DNAfragments into PER.C6 cells: pAd5/S1800-HSA digested with EcoRI and SalI(2 μg). At occurrence of CPE, the virus was harvested and amplified byserial passages of PER.C6 cells. The activity of this HSA-luc virus wascompared to single insert ΔE1 viruses containing either the S1800-HSA orthe CMV-luc transcription units in the E1 region. A549 cells were seededat 2×10⁵ cells per well and infected 5 hrs later with different amountsof the virus. Two days later, transgene expression was measured.Luciferase activity was measured using a luciferase assay system(Promega) and expression of the murine HSA gene was measured with anα-HSA antibody (M1/69, Pharmingen). The results are listed in Table III.

This experiment shows that using the plasmid-based recombination system,double insert viruses can be made and that both inserts are functional.Furthermore, the luciferase activity of the double insert viruses iscomparable to the CMV-driven luciferase activity of the control virus.Therefore, the E3 promoter was concluded to be highly active in A549cells, even in the absence of E1A proteins.

In addition to manipulations in the E3 region, changes of the E4 regionor parts of the E4 region can be accomplished easily in pBr/Ad.Bam-rITR.Generation and propagation of such a virus, however, in some casesdemands complementation in trans.

Example 3 Demonstration of the Competence of a Synthetic DNA Sequence,which is Capable of Forming a Hairpin Structure, to Serve as a Primerfor Reverse Strand Synthesis for the Generation of Double-stranded DNAMolecules in Cells that Contain and Express Adenoviral Genes

Name convention of the plasmids used:

p plasmid I ITR (Adenoviral ITR) C Cytomegalovirus (CMV)Enhancer/Promoter Combination L Firefly Luciferase Coding Sequence hac,haw Potential hairpin that can be formed after digestion withrestriction endonuclease Asp718 in both the correct and in the reverseorientation, respectively (FIG. 15)

The naming convention is exemplified as follows. pICLhaw is a plasmidthat contains the adenoviral ITR followed by the CMV-driven luciferasegene and the Asp718 hairpin in the reverse (non-functional) orientation.

Plasmids plCLhac, pICLhaw, pICLI, and pICL were generated using standardtechniques. The schematic representation of these plasmids is shown inFIGS. 16–19.

Plasmid pICL is derived from the following plasmids:

nt.1 457 pMLP10 (Levrero et al, (1991) Gene 101: 195–202) nt.458 1218pCMVβ (Clontech, EMBL Bank No. U02451) nt.1219 3016 pMLP.luc (IntroGene,unpublished) nt.3017 5620 pBLCAT5 (Stein et al, (1989) Mol. Cell Biol.9: 4531–4).

The plasmid has been constructed as follows:

The tet gene of plasmid pMLP10 has been inactivated by deletion of theBamHI-SalI fragment, to generate pBLP10ΔSB. Using primer set PCR/MLP1(SEQ ID NO:37) and PCR/MLP3 (SEQ ID NO:38), a 210 bp fragment containingthe Ad5-ITR, flanked by a synthetic SalI restriction site, was amplifiedusing pMLP10 DNA as the template. The PCR product was digested with theenzymes EcoRI and SgrAI to generate a 196 bp fragment. Plasmid pMLP10ΔSBwas digested with EcoRI and SgrAI to remove the ITR. This fragment wasreplaced by the EcoRI-SgrAl-reated PCR fragment to generate pMLP/SAL.

Plasmid pCMV-Luc was digested to completion with PvuII and recirculatedto remove the SV40-derived poly-adenylation signal and Ad5 sequences,with the exception of the Ad5 left-terminus. In the resulting plasmid,pCMV-lucΔAd, the Ad5 ITR was replaced by the Sal-site-flanked ITR fromplasmid pMLP/SAL by exchanging the XmnI-SacII fragments. The resultingplasmid, pCMV-lucΔAd/SAL, the Ad5 left terminus, and the CMV-drivenluciferase gene were isolated as a SalI-SmaI fragment and inserted intothe SalI and HpaI digested plasmid pBLCATS, to form plasmid pICL.Plasmid pICL is represented in FIG. 19 and its sequence is presented inFIG. 20.

Plasmid pICL contains the following features:

nt.1–457 Ad5 left terminus (Sequence 1-457 of human adenivorus type 5)nt.458–969 Human cytomegalovirus enhancer and immediate early promoter(Boshart et al, (1985) Cell 41: 521– 530) (from plasmid pCMVβ, Clontech,Palo Alto, USA) nt.970–1204 SV40 19S exon and truncated 16/19S intron(from plasmid pCMVβ) nt.1218–2987 Firefly luciferase gene (frompMLP.luc) nt.3018–3131 SV40 tandem poly-adenylation signals from latetranscript, derived from plasmid pBLCAT5) nt.3132–5620 pUC12 backbone(derived from plasmid pBLCAT5) nt.4337–5191 β-lactamase gene(Amp-resistance gene, reverse orientation)Plasmids pICLhac and pICLhaw

Plasmids pICLhac and pICLhaw were derived from plasmid pICL by digestionof pICL with the restriction enzyme Asp718. The linearized plasmid wastreated with Calf-Intestine Alkaline Phosphatase to remove the 51phosphate groups. The partially complementary synthetic single-strandedoligonucleotides Hp/asp1 (SEQ ID NO:39) and Hp/asp2 (SEQ ID NO:40) wereannealed and phosphorylated on their 5′ ends using T4-polynucleotidekinase.

The phosphorylated double-stranded oligomers were mixed with thedephosphorylated pICL fragment and ligated. Clones containing a singlecopy of the synthetic oligonucleotide inserted into the plasmid wereisolated and characterized using restriction enzyme digests. Insertionof the oligonucleotide into the Asp718 site will at one junctionrecreate an Asp718 recognition site, whereas at the other junction therecognition site will be disrupted. The orientation and the integrity ofthe inserted oligonucleotide were verified in selected clones bysequence analyses. A clone containing the oligonucleotide in the correctorientation (the Asp718 site close to the 3205 EcoRI site) was denotedpICLhac. A clone with the oligonucleotide in the reverse orientation(the Asp718 site close to the SV40 derived poly signal) was designatedpICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and17.

Plasmid pICLI was created from plasmid pICL by insertion of theSalI-SgrAI fragment from pICL, containing the Ad5-ITR, into the Asp718site of pICL. The 194 bp SalI-SgrAI fragment was isolated from pICL andthe cohesive ends were converted to blunt ends using E. coli DNApolymerase I (Klenow fragment) and dNTP's. The Asp718 cohesive ends wereconverted to blunt ends by treatment with mungbean nuclease. Clones thatcontained the ITR in the Asp718 site of plasmid pICL were generated byligation. A clone that contained the ITR fragment in the correctorientation was designated pICLI (FIG. 18).

Recombinant adenovirus Ad-CMV-hcTK was constructed according to themethod described in Patent application 95202213. Two components arerequired to generate a recombinant adenovirus. First, anadaptor-plasmid, which contains the left terminus of the adenoviralgenome containing the ITR and the packaging signal, an expressioncassette with the gene of interest, and a portion of the adenoviralgenome which can be used for homologous recombination, is necessary. Inaddition, adenoviral DNA is needed for recombination with theaforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, the plasmidpCMV.TK was used as a basis. This plasmid contains nt.1–455 of theadenovirus type 5 genome, nt. 456–1204 derived from pCMVβ (Clontech, thePstI-StuI fragment that contains the CMV enhancer promoter and the16S/19S intron from simian Virus 40), the Herpes Simplex Virus thymidinekinase gene (described in EP patent application 95202213.5), theSV40-derived polyadenylation signal (nt. 2533–2668 of the SV40sequence), and the BglII-ScaI fragment of Ad5 (nt. 3328–6092 of the Ad5sequence). These fragments are present in a pMLP10-derived (Levrero etal, (1991) Gene 101: 195–202) backbone. To generate plasmidpAD-CMVhc-TK, plasmid pCMV.TK was digested with ClaI (the uniqueClaI-site is located just upstream of the TK open reading frame) anddephosphorylated with Calf-Intestine Alkaline Phosphate. To generate ahairpin-structure, the synthetic oligonucleotides HP/cla1 (SEQ ID NO:41)and HP/cla2 (SEQ ID NO:42) were annealed and phosphorylated on their5′-OH groups with T4-polynucleotide kinase and ATP. The double-strandedoligonucleotide was ligated with the linearized vector fragment and usedto transform E. coli strain Sure. Insertion of the oligonucleotide intothe ClaI site will disrupt the ClaI recognition sites. Theoligonucleotide contains a new ClaI site near one of its termini. Inselected clones, the orientation and the integrity of the insertedoligonucleotide were verified by sequence analyses. A clone containingthe oligonucleotide in the correct orientation (the ClaI site at the ITRside) was denoted pAd-CMV-hcTK. This plasmid was co-transfected withClaI-digested wild-type adenovirus-type5 DNA into 911 cells. Arecombinant adenovirus in which the CMV-hcTK expression cassettereplaced the E1 sequences was isolated and propagated using standardprocedures.

To study whether the hairpin can be used as a primer for reverse strandsynthesis on the displaced strand after replication has started at theITR, the plasmid pICLhac was introduced into 911 cells. The plasmidpICLhaw served as a control: it contains the oligonucleotide pair HP/asp1 (SEQ ID NO:39) and 2 (SEQ ID NO:40) in the reverse orientation but isotherwise identical to plasmid pICLhac. Also included in these studieswere plasmids pICLI and pICL. In pICLI, the hairpin is replaced by anadenoviral ITR. pICL contains neither a hairpin nor an ITR sequence.These plasmids served as controls to determine the efficiency ofreplication by virtue of the terminal hairpin structure. To provide theviral products other than the E1 proteins (these are produced by the 911cells) required for DNA replication, the cultures were infected with thevirus IG.Ad.MLPI.TK after transfection. Several parameters were studiedto demonstrate proper replication of the transfected DNA molecules.First, DNA extracted from the cell cultures transfected with theaforementioned plasmids and infected with IG.Ad.MLPI.TK virus wasanalyzed by Southern blotting for the presence of the expectedreplication intermediates, as well as for the presence of the duplicatedgenomes. Furthermore, from the transfected and IG.Ad.MLPI.TK infectedcell populations, virus was isolated that could transfer a luciferasemarker gene into luciferase negative cells and express the gene.

Plasmid DNA of pICLhac, pCLhaw, pICLI, and pICL were digested withrestriction endonuclease SalI and treated with mungbean nuclease toremove the four nucleotide single-stranded extension of the resultingDNA fragment. In this manner, a natural adenoviral 5′ ITR terminus onthe DNA fragment was created. Subsequently, both pICLhac and pICLhawwere digested with restriction endonuclease Asp718 to generate theterminus capable of forming a hairpin structure. The digested plasmidswere introduced into 911 cells, using the standard calcium phosphateco-precipitation technique. Four dishes for each plasmid were prepared.During the transfection, two of the cultures of each plasmid wereinfected with the IG.Ad.MLPI.TK virus using five infectiousIG.Ad.MLPI.TK particles per cell. At twenty-hours post transfection andforty hours post-transfection, one Ad.TK-virus-infected and oneuninfected culture were used to isolate low molecular-weight DNA usingthe procedure devised by Hirt (as described in Einerhand et al, (1995)Gene Therapy 2:336–343). Aliquots of isolated DNA were used for Southernanalysis. After digestion of the samples with restriction endonucleaseEcoRI using the luciferase gene as a probe, a hybridizing fragment ofapproximately 2.6 kb was only detected in samples from theadenoviral-infected cells transfected with plasmid pICLhac. The size ofthis fragment was consistent with the anticipated duplication of theluciferase marker gene. This supports the conclusion that the insertedhairpin is capable of serving as a primer for reverse strand synthesis.The hybridizing fragment would be absent if the IG.Ad.MLPI.TK virus wasomitted or if the hairpin oligonucleotide was inserted in the reverseorientation.

The restriction endoculease DpnI recognizes the tetranucleotide sequence5′-GATC-3′ but cleaves only methylated DNA (plasmid DNA propagated in,and derived from, E. coli, not DNA that has been replicated in mammaliancells). The restriction endonuclease MboI recognizes the same sequencesbut cleaves only unmethylated DNA (DNA propagated in mammalian cells).DNA samples isolated from the transfected cells are incubated with MboIand DpnI and analyzed with Southern blots. These results demonstratedthat large DpnI-resistant fragments, which were absent in the MboItreated samples, were present only in the cells transfected with pICLhacand the pICLI. These data demonstrate that replication and duplicationof the fragments occur only after transfection of pICLI and pICLhac.

These data demonstrate that in adenoviral-infected cells, linear DNAfragments that have an adenoviral-derived ITR at one terminus and anucleotide sequence that can anneal to sequences on the same strand atthe other terminus, when present in single-stranded form, generate ahairpin structure and will be converted to structures that have ITRsequences on both ends. The resulting DNA molecules will replicate bythe same mechanism as the wild-type adenoviral genomes.

Example 4 Demonstration that the DNA Molecules that Contain a LuciferaseMarker Gene, a Single Copy of the ITR, the Encapsidation Signal, and aSynthetic DNA Sequence that is Capable of Forming a Hairpin Structureare Sufficient to Generate DNA Molecules that Can Be Encapsidated intoVirions

To demonstrate that the DNA molecules generated in Example 3, whichcontain two copies of the CMV-luc marker gene, can be encapsidated intovirions, virus was harvested from the remaining two cultures via threecycles of freeze/thaw crushing and was used to infect murinefibroblasts. Forty-eight hours after infection, the infected cells wereassayed for luciferase activity. To exclude the possibility that theluciferase activity had been induced by transfer of free DNA rather thanby virus particles, virus stocks were treated with DNaseI to remove DNAcontaminants. Furthermore, as an additional control, aliquots of thevirus stocks were incubated for 60 minutes at 56° C. The heat treatmentdoes not affect the contaminating DNA but does inactivate the viruses.Significant luciferase activity was only found in the cells infectedwith the virus stocks derived from IG.Ad.MLPI.TK-infected cellstransfected with pICLhc and pICLI. No significant luciferase activitywas found in the non-infected cells or in the infected cells transfectedwith pICLhw and pICL. Heat inactivation, but not DNaseI treatment,completely eliminated luciferase expression, demonstrating thatadenoviral particles, but not free (contaminating) DNA fragments, wereresponsible for transfer of the luciferase reporter gene.

The results demonstrate that these small viral genomes can beencapsidated into adenoviral particles and suggest that the ITR and theencapsidation signal are sufficient for encapsidation of linear DNAfragments into adenoviral particles. These adenoviral particles can beused for efficient gene transfer. When introduced into cells thatcontain and express at least some of the adenoviral genes (namely E1,E2, E4, and L, and VA), recombinant DNA molecules that include at leastone ITR, at least part of the encapsidation signal, and a synthetic DNAsequence that is capable of forming a hairpin structure have theintrinsic capacity to autonomously generate recombinant genomes that canbe encapsidated into virions. Such genomes and vector systems can beused for gene transfer.

Example 5 Demonstration that DNA Molecules, which Contain Nucleotides3510–35953 of the Adenovirus Type 5 Genome and a Terminal DNA Sequencethat is Capable of Forming a Hairpin Structure, Can Replicate in 911Cells

In order to develop a replicating DNA molecule that can provide theadenoviral products necessary to allow the ICLhac vector genome andsimilar minimal adenovectors to be encapsidated into adenoviralparticles by helper cells, the Ad-CMV-hcTK adenoviral vector wasdeveloped. The annealed oligonucleotide pair (Table I) HP/cla 1 and 2was inserted between the CMV enhancer/promoter region and the thymidinekinase gene. The vector Ad-CMV-hcTK was propagated and produced in 911cell using standard procedures. This vector was grown and propagatedexclusively as a source of DNA used for transfection. DNA of theadenoviral Ad-CMV-hcTK was isolated from viral particles that had beenpurified using CsCl density-gradient centrifugation by standardtechniques. The viral DNA was digested with restriction endonucleaseClaI. The digested DNA was size-fractionated on a 0.7% agarose gel andthe large fragment was isolated and used for further experiments.Cultures of 911 cells were transfected with the large ClaI-fragment ofthe Ad-CMV-hcTK DNA using standard calcium phosphate co-precipitationtechniques. Similar to the previous experiments with pICLhac, theAd-CMV-hc replicates starting at the right-hand ITR. Once the 1-strandis displaced, a hairpin can be formed at the left-hand terminus of thefragment. This facilitates DNA polymerase elongation of the chaintowards the right-hand side. The process proceeds until the displacedstrand is completely converted to its double-stranded form. Finally, theright-hand ITR is recreated and normal adenoviral replication-initiationand elongation occur at this location. The polymerase reads through thehairpin, thereby duplicating the molecule. The input DNA molecule of33250 bp, which had an adenoviral ITR sequence at one terminus and a DNAsequence that had the capacity to form a hairpin structure on the otherterminus, is duplicated so that both ends contain an ITR sequence. Theresulting DNA molecule consists of a palindromic structure ofapproximately 66500 bp.

This structure is detected in low-molecular weight DNA extracted fromtransfected cells using Southern analysis. The palindromic nature of theDNA fragment can be demonstrated by digestion of the low-molecularweight DNA with suitable restriction endonucleases and Southern blottingwith the HSV-TK gene as the probe. This molecule can self-replicate inthe transfected cells by virtue of the adenoviral gene products that arepresent in the cells. In part, the adenoviral genes are expressed fromtemplates that are integrated in the genome of the target cells (namely,the E1 gene products), while the other genes reside in the replicatingDNA fragment itself. This linear DNA fragment cannot be encapsidatedinto virions. Not only does it lack all the DNA sequences required forencapsidation, but its size is much too large to be encapsidated.

Example 6 Demonstration that DNA Molecules, which Contain Nucleotides3503–35953 of the Adenovirus Type 5 Genome and a Terminal DNA Sequencethat is Capable of Forming a Hairpin Structure, Can Replicate in 911Cells and Can Provide the Helper Functions Required to Encapsidate thepICLI and pICLhac Derived DNA Fragments

The purpose of the next series of experiments is to demonstrate that theDNA molecule described in Example 5 can be used to encapsidate theminimal adenovectors described in Examples 3 and 4.

The large fragment isolated after endonuclease ClaI-digestion ofAd-CMV-hcTK DNA was introduced into 911 cells (as described in Example5) along with endonuclease SalI, mungbean nuclease, endonucleaseAsp718-treated plasmid pICLhac, or as a control similarly treatedplasmid pICLhaw. After 48 hours, virus was isolated by freeze/thawcrushing of the transfected cell population. The virus preparation wastreated with DNaseI to remove contaminating free DNA. The virus was usedsubsequently to infect Rat2 fibroblasts. Forty-eight hours postinfection, the cells were assayed for luciferase activity. Significantluciferase activity was only demonstrated in the cells infected withvirus isolated from the cells transfected with pICLhac. No activity wasdemonstrated in the cells infected with virus isolated from the cellstransfected with pICLhaw. Heat inactivation of the virus prior toinfection completely abolished the luciferase activity, indicating thatthe luciferase gene was transferred by a viral particle. Infection of911 cells with the virus stock did not result in any cytopathologicaleffects, demonstrating that pICLhac was produced without any infectioushelper virus being propagated on 911 cells. These results demonstratethat the proposed method can be used to produce stocks ofminimal-adenoviral vectors that are completely devoid of infectioushelper viruses that are able to replicate autonomously onadenoviral-transformed human cells or on non-adenoviral transformedhuman cells.

Example 7 Construction of Plasmids for the Generation and Production ofMinimal Adenoviral Vectors

A minimal adenoviral vector contains, as operably linked components, theadenoviral-derived cis elements necessary for replication and packaging,with or without foreign nucleic acid molecules to be transferred.Recently, the lower limit for efficient packaging of adenoviral vectorshas been determined at 75% of the genome length (Parks and Graham,1997). To allow flexible incorporation of various lengths of stufferfragments, a multiple cloning site (MCS) was introduced into a minimaladenoviral vector. To obtain a minimal adenoviral vector according tothe invention, the following constructs were made: pAd/L420-HSA (FIG.19) was digested with BglII and SalI and the vector-containing fragmentwas isolated. This fragment contains the left ITR and packaging signalfrom Ad5 and the murine HSA gene driven by a modified retroviral LTR.The right adenoviral ITR was amplified by PCR on a pBr/Ad.BamHI-rITRtemplate DNA using the following primers: PolyL-ITR: 5′-AAC-TGC-AGA-TCT-ATC-GAT-ACT-AGT-CAA-TTG-CTC-GAG-TCT-AGA-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3′(SEQ ID NO:14) and ITR-BSN:5′-CGG-GAT-CCG-TCG-ACG-CGG-CCG-CAT-CAT-CAA-TAA-TAT-ACC-3′ (SEQ IDNO:15). The amplified fragment was digested with PstI and BamHI andcloned into pUC119 digested with the same enzymes. After sequenceconfirmation of correct amplification of the ITR and the MCS, aBgllI-SalI fragment was isolated and cloned into the BglII/SalI-digestedpAd/L420-HSA fragment described above. The resulting clone was namedpAd/L420-HSA.ITR.

To be able to manipulate constructs of lengths exceeding 30 kb, theminimal adenoviral vector pAd/L420-HSA.ITR was subcloned in a cosmidvector background. For this purpose, the cosmid vector pWE15 wasmodified to remove restriction sites in the backbone. pWE15 was digestedwith PstI and fragments of 4 kb and 2.36 kb were isolated from anagarose gel and ligated together. The resulting clone, stripped of theSV40 ori/early promoter and neomycine resistance coding sequence, wasnamed pWE20. Then, pWE20 was digested with ClaI and HindIII and thesticky ends were filled in with Klenow enzyme. A 6354 bp blunt fragmentwas ligated to a phosphorylated NsiI linker with the following sequence:5′-CGATGCATCG-3′ (SEQ ID NO:16). The ligated DNA was phenol/chloroformextracted, precipitated with EtOH to change buffers, and digested withexcess NsiI. Digested DNA was separated from the linkers byelectrophoresis, isolated, and then religated. The resulting clone wasnamed pWE25. Correct insertion of the NsiI linker was confirmed byrestriction enzyme digestion and sequencing. To construct the minimaladenoviral vector, pAd/L420-HSA.ITR was digested with ScaI and NotI. Theresulting 2 kb fragment, containing part of the ampicillin gene and theadeno ITRs, was cloned into pWE25 digested with ScaI and NotI. Theresulting clone was named pMV/L420H (FIG. 24). This clone allows easymanipulation to exchange the promoter and/or gene and also allowsinsertion of DNA fragments of not-easily-cloned lengths into normalplasmid backbones.

Plasmid pMV/CMV-LacZ was made by exchanging the L420-HSA fragment(SnaBI-BamHI) for a fragment from pcDNA3-nlsLacZ (NruI-BamHI) thatcontains the CMV promoter and LacZ coding sequences. pcDNA3-nlsLacZ wasconstructed by insertion of a KpnI-BamHI fragment obtained after PCRamplification of the nlsLacZ coding sequences into pcDNA3 (Invitrogen)digested with KpnI and BamHI. The PCR reaction was performed on apMLP.nlsLacZ template DNA using the primers 1:5′-GGG-GTG-GCC-AGG-GTA-CCT-CTA-GGC-TTT-TGC-AA-3′ (SEQ ID NO:17) and 2:5′-GGG-GGG-ATC-CAT-AAA-CAA-GTT-CAG-AAT-CC-3′ (SEQ ID NO:18). Correctamplification and cloning were confirmed by assaying P-galactosidaseexpression in a transient transfection experiment in 911 cells.

The vector pAd/MLPnlsLacZ was made as follows: pMLP10 (Levrero et al,(1991) Gene 101: 195–202) was digested with HindIII and BamHI andligated, in a three-part ligation, to a 3.3 kb AvrII-BamHI fragment fromL7RHbgal (Kalderon et al, (1984) Cell 499–509), and a synthetic linkerwith HindIII and XbaI overhang. The linker was made by annealing twooligonucleotides of sequence 5′-AGC TTG AAT TCC CGG GTA CCT-3′ (SEQ IDNO:19) and 5′-CTA GAG GTA CCC GGG AAT TCA-3′ (SEQ ID NO:20). Theresulting clone was named pMLP.nlsLacZ/-Ad. Next, pMLP.nlsLacZ/-Ad wasdigested with BamHI and NruI and the vector containing fragment wasligated to a 2766 bp BglII-ScaI fragment from pAd5SalB (Bernards et al,(1982) Virology 120:422–432). This resulted in the adapter plasmidpMLP.nlsLacZ (described in EP 0 707 071).

Propagation of a minimal adenoviral vector can only be achieved byexpression of adenoviral gene products. Expression of adenoviral geneproducts at levels high enough to sustain production of large quantitiesof virus requires replication of the coding nucleic acid molecule.Usually, replicating helper viruses are used to complement the minimaladenoviral vectors. However, the present invention provides packagingsystems for minimal adenoviral vectors without the use of helperviruses. One of the methods of the invention makes use of a replicatingDNA molecule that contains the 5′-ITR and all adenoviral sequencesbetween bp 3510 and 35938, i.e., the complete adenoviral genome exceptfor the E1 region and the packaging signal. Construct pWE/Ad.Δ5′ (FIG.23) is an example of a replicating molecule according to the inventionthat contains two adenoviral ITRs. pWE/Ad.Δ5′ as made in a cosmid vectorbackground from three fragments. First, the 5′ ITR from Ad5 wasamplified using the following primers: ITR-EPH:5′-CGG-AAT-TCT-TAA-TTA-AGT-TAA-CAT-CAT-CAA-TAA-TAT-ACC-3′ (SEQ ID NO:21)and ITR-pIX:5′-ACG-GCG-CGC-CTT-AAG-CCA-CGC-CCA-CAC-ATT-TCA-GTA-CGT-ACT-AGT-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3′(SEQ ID NO:22). The resulting PCR fragment was digested with EcoRI andAscI and cloned into vector pNEB193 (New England Biolabs) digested withthe same enzymes. The resulting construct was named pNEB/ITR-pIX.Sequencing confirmed correct amplification of the Ad5 sequences in theleft ITR (Ad5 sequences 1 to 103) linked to the pIX promoter (Ad5sequences 3511 to 3538), except for a single mismatch with the expectedsequence according to GenBank (Accession no.: M73260/M29978), i.e., anextra C residue was found just upstream of the AflII site. This ITR-pIXfragment was isolated with EcoRI and AflII and ligated to an EcoRI-AflIIvector fragment containing Ad5 sequences 3539–21567. The latter fragmentwas obtained by digestion of pBr/Ad.Cla-Bam (supra) with EcoRI andpartially with AflII. The resulting clone was named pAd/LITR(Δ5′)-BamHI.The final construct pWE/Ad.Δ5′ was made by ligating cosmid vectorpWE15.Pac (supra) digested with PacI to pAd/LITR(Δ5′)-BamHI digestedwith PacI/BamHI and pBr/Ad.Bam-rITR.pac#2 (supra) digested withPacI/BamHI (FIG. 23).

An alternative method to produce packaging systems for minimaladenoviral vectors without the use of helper viruses according to theinvention is to use a replicating DNA molecule. The replicating DNAmolecule must contain the complete adenoviral genome, except for the E1region and the packaging signal, and also one of the ITRs in themolecule must be replaced by a fragment containing a DNA sequencecomplementary to a portion of the same strand other than the ITR so thatthe molecule is able to form a hairpin structure (FIG. 10). In anon-limiting example, the DNA sequence complementary to a portion of thesame strand other than the ITR is derived from the adeno-associatedvirus (AAV) terminal repeat. Such a replicating DNA molecule is madefollowing the same cloning strategy as described for pWE/Ad.Δ5′, exceptthat the AAV terminal repeat is linked to part of the adenoviral pIXpromoter. To this end, the adenoviral ITR sequences between the HpaI andSpeI sites in construct pNEB/ITR-pIX were exchanged for the AAV ITR byintroducing the PvuII/XbaI fragment from psub201(+) containing the AAVITR (Samulski et al, (1989) J. Virol. 63:3822–3828). This results inconstruct pWE/AAV.Δ5′ that replicates in an E1 complementing cell line.

Another alternative packaging system for minimal adenoviral vectors isdescribed infra and makes use of the replication system of SV40. Afunctional helper molecule according to this method contains at leastthe adenoviral sequences necessary to sustain packaging of a minimalconstruct, but does not contain the E1 sequences and packaging signaland preferably also lacks ITRs. This adenoviral-derived entity has to bepresent on a vector that contains, besides the sequences needed forpropagation in bacteria, an origin of replication from SV40 virus.Transfection of this molecule, along with the minimal adenoviral vectordescribed supra, into a packaging cell line (e.g., PER.C6) expressing,besides the E1 proteins, SV40 derived Large T antigen proteins, resultsin Large T-dependent replication of the adenoviral-derived helperconstruct. This replication leads to high levels of adenoviral proteinsnecessary for replication of the minimal adenoviral vector and packaginginto virus particles. In this way, there is no sequence overlap thatleads to homologous recombination between the minimal adenoviral vectorconstruct and the helper molecule. In addition, there is no sequenceoverlap that leads to homologous recombination between the helpermolecule and minimal adenoviral vector on the one side and the E1sequence in the packaging cell on the other side.

Replication of a 40 kb adenoviral construct was investigated in cellsexpressing SV40 Large T proteins. Cells (2×10⁶ Cos-1) were transfectedin a T25 flask with the following constructs complexed withlipofectamine reagent (Life techn.): the 8 kb cosmid vector pWE.pac, the40.5 kb construct pWE/Ad.AflII-rITR, and three clones (#1, #5 and #9) ofthe 40.6 kb construct pWE/Ad.Δ5′ (described infra). Controltransfections were carried out with the constructs pWE.pac andpWE/Ad.AflII-rITR digested with PacI enzyme and a CMV-LacZ expressionvector without the SV40 ori sequence. Transfection efficiency was 50% asdetermined by a separate transfection using the CMV-LacZ vector andX-gal staining after 48 hrs. All cells were harvested 48 hrs. aftertransfection and DNA was extracted according to the Hirt procedure (asdescribed in Einerhand et al, (1995) Gene Therapy 2:336–343). Finalpellets were resuspended in 50 μl TE+RNase (20 μg/ml) and 10 μl sampleswere digested with MboI (35 units overnight at 37° C.). Undigestedsamples (5 μl) and MboI digested samples were run on a 0.8% agarose gel,transferred to a nylon filter (Amersham), and hybridized to radioactiveprobes according to standard procedures. One probe was derived from an887 bp DpnI fragment from the cosmid vector pWE.pac; the other probe wasderived from a 1864 bp BsrGI-BamHI fragment from adenoviral sequences.These probes hybridize to an 887 bp band and a 1416 bp band,respectively, in MboI digested material. Input DNA from bacterial originis methylated and therefore not digested with MboI. Therefore, it ispossible to specifically detect DNA that is replicated in eukaryoticcells. FIG. 26A shows a schematic presentation of the constructpWE/Ad.Δ5′ and also shows the locations of the SV40 origin ofreplication, the pWE-derived probe, and the adenoviral-derived probe.The lower part of the FIG. presents the autoradiograms of the Southernblots hybridized to the adenoviral probe (B) and the pWE probe (C). Seelegends for explanation of sample loading. These experiments show thatall lanes that contain material from Cos-1 cells that were transfectedwith plasmids harbouring an SV40 ori sequence contain MboI sensitive DNAand show a specific band of the expected length. The bands specific forreplication in the lanes with Cos-1 cells transfected with PacI digestedmaterial (lanes B 17/18 and C 15–18) probably result from incompletePacI digestion. From these experiments it can be concluded that largeDNA fragments can be replicated with the SV40 LargeT/ori system ineukaryotic cells.

Example 8

A functional adenoviral helper molecule lacking ITR sequences wasconstructed starting with the clone pWE/Ad.D5′ described supra.pWE/Ad.D5′ was digested with Bst1107I and the 17.5 kb vector-containingfragment was religated to give pWE/Ad.D5′-Bst1107I. This clone was thenused to amplify the 3′ part of the adenoviral genome sequences withoutthe right ITR. A 2645 bp PCR fragment was generated using the primersAd3′/Forw: 5′-CGG AAT TCA TCA GGA TAG GGC GGT GG-3′ (SEQ ID NO:23) andAd3′/Rev: 5′-CGG GAT CCT ATC GAT ATT TAA ATG TTT TAG GGC GGA GTA ACTTG-3′ (SEQ ID NO:24). The amplified fragment was digested with EcoRI andBamHI and subcloned into pBr322 digested with the same enzymes. Afterconfirmation of correct amplification by sequencing, the 2558 bpSbfI-ClaI fragment of the clone was recloned into pWE/Ad.D5′-Bst1107Idigested with the same enzymes. The resulting construct lacks the rightITR and is named pWE/ΔrI-Bst1107I. Next, the left ITR was replaced by alinker with a PacI and AflII overhang constructed by annealing thefollowing primers: PA-pIX1 5′-TAA GCC ACT AGT ACG TAC TGA AAT GTG TGGGCG TGG C-3′ (SEQ ID NO:25) and PA-pIX2 5′-TTA AGC CAC GCC CAC ACA TTTCAG TAC GTA CTA GTG GCT TAA T-3′ (SEQ ID NO:26). The removal of the leftITR restored correct sequence of the pIX promoter. This clone is namedpWE/ΔITR-Bst1107I. Correct insertion of the double stranded linker wasconfirmed by sequencing. The deleted Bst1107I fragment was then clonedback into pWE/ΔITR-Bstl 1107I and the correct orientation was checked byrestriction digestion. The resulting clone is named pWE/Ad-H. Followingtransfection of this DNA molecule into packaging cells that expressadenoviral E1 proteins and the SV40 Large T antigen, replication of themolecule takes place, resulting in high levels of adenoviral proteinsencoded by the adenoviral entity on that molecule.

Example 9 Miniaturized, Multiwell Production of Recombinant AdenoviralVectors

A 96-well microtiter tissue culture plate (plate 1) (Greiner, NL,catalogue #6555180) was coated with poly-L-lysine (PLL, 0.1 mg/ml)(Sigma) dissolved in sterile water by incubating each well for 20–120minutes at room temperature. Alternatively, precoated 96-well plates canbe used (Becton Dickinson). After the incubation with PLL, each well waswashed two times with 100 μl sterile water and dried at room temperaturefor at least two hours. The day before transfection, PER.C6 cells wereharvested using trypsin-EDTA and counted. The cells were then diluted toa suspension of 45,000 cells per 100 μl, followed by seeding 100 μl ofcell suspension per well in the PLL coated 96-well plates. The next day,2.6 μl of SalI linearized pAd/CMV-LacZ (1 μg/μl), 2.6 μl of PacIlinearized pWE-Ad.AflII-rITR plasmid DNA (1 μg/μl), and 95 μl serum freeDulbecco's Modified Eagles Medium (DMEM) were mixed with 25.6 μllipofectamine diluted in 74.4 μl serum free DMEM by adding thelipofectamine to the DNA mix. The DNA/lipofectamine mixture was left atroom temperature for 30 minutes after which 1.3 ml serum free medium wasadded. The latter mixture was then added (30 μl per well) to PER.C6seeded wells that were washed with 200 μl DMEM prior to transfection.After 3 hours in a humidified CO₂ incubator (37° C., 10% CO₂), 200 μlDMEM with 10% fetal calf serum and 10 mM MgCl₂ was added to each welland the plates were returned to the humidified CO₂ incubator (37° C.,10% CO₂). The next day, the medium of each well was replaced with 200 μlDMEM, 10% FCS, 10 mM MgCl₂. The plates were then left in the humidifiedCO₂ incubator for an additional three days, after which the wells weresubjected to freezing at −20° C. for at least 1 hour followed by thawingand resuspension by repeated pipetting. Transfection efficiency wasdetermined using lacZ staining in additional plates and found to beapproximately 40% for each transfected well of PER.C6 cells. An aliquotof 100 μl of freeze/thawed transfected cells was transferred to eachwell of a plate with new PER.C6 cells seeded as described above, exceptwithout PLL coated plates (plate 2). The second 96-well plate waschecked for CPE. At least 5% of the wells showed clear CPE after 2 days.Four days after infection with the lysate from plate 1, the plate wassubjected to one freeze/thaw cycle and 10 μl from each lysed well wasadded to wells of a plate seeded with A549 cells (1×10⁴ cells per wellseeded in 100 μl in DMEM, 10% FCS the day before). Two days afterinfection, the wells were stained for lacZ activity. Of the infectedwells, 96% were infected and stained blue. All wells stained and a largenumber of wells showed 100% blue staining implying transduction of allcells with adenoviral vector carrying lacZ. The adenoviral titer ofwell-produced virus is around 10⁶–10⁷ infectious units per ml asdetermined by extrapolation from MOI experiments in tissue cultureflasks.

The subject invention discloses methods and compositions for the highthroughput delivery and expression in a host of sample nucleic acid(s)encoding product(s) of unknown function. Methods are described for theconstruction of complementing cell lines, libraries of adenoviralderived plasmids containing sample nucleic acids, packaging theadenoviral-derived plasmids into adenoviral vectors, infecting a hostwith the adenoviral vectors that express the product(s) of the samplenucleic acid(s) in the host, identifying an altered phenotype induced inthe host by the product(s) of the sample nucleic acids, and therebyassigning a function to the product(s) encoded by the sample nucleicacids. The sample nucleic acids can be, for example, syntheticoligonucleotides, DNAs, or cDNAs and can encode, for example,polypeptides, antisense nucleic acids, or GSEs. The methods can be fullyautomated and performed in a multiwell format to allow for convenienthigh throughput analysis of sample nucleic acid libraries.

Example 10 Miniaturized, Multiwell Production of E1 and E2A DeletedRecombinant Adenoviral Vectors Carrying Therapeutic and MarkerTransgenes

To allow the construction of cDNA libraries with a representativerepertoire of cDNA sequences, the cloning capacity of the miniaturizedadenoviral production system PER.C6/E2A, a derivative of PER.C6, wasused. This cell line allows the production of a vector with threedeletions of adenoviral expression cassettes: E1, E2A, and E3. Thesethree deletions allow the theoretical cloning of vectors with transgenesizes of up to about 10.5 kb in length. The production of E1 and E2Adeleted vectors carrying a variety of human and mouse cDNAs, as well asadditional marker genes, is shown.

The day before transfection, PER.C6/E2A cells were harvested usingtrypsin-EDTA and counted. The cells were then diluted with culturemedium (DMEM with 10% fetal bovine serum and 10 mM MgCl₂) to asuspension of 22,500 cells per 100 μl followed by seeding 100 μl perwell in poly-L-lysine (PLL) coated 96-well plates (Becton Dickinson).The next day, 2.6 μg of the linearized adapter molecules and 2.6 μg ofPacI linearized pWE/Ad.AflII-rITR.deltaE2A plasmid DNA (see example 19)in 100 μl serum free Dulbecco's Modified Eagles Medium (DMEM) were mixedwith 25.6 μl lipofectamine diluted in 74.4 μl serum free DMEM by addingthe lipofectamine mixture to the DNA mix. The DNA/lipofectamine mixturewas left at room temperature for 30 minutes, after which 1.3 ml serumfree medium was added. The latter mixture (30 μl per well) was thenadded to PER.C6/E2A seeded wells that were washed with 200 μl DMEM priorto transfection. After 3 hours in a humidified CO₂ incubator (39° C.,10% CO₂), 200 μl DMEM with 10% fetal bovine serum and 10 MM MgCl₂ wasadded to each well and the plates were returned to the humidified CO₂incubator (39° C., 10% CO₂). The next day, the medium of each well wasreplaced with 200 μl DMEM with 10% fetal bovine serum and 10 mM MgCl₂.The plates were then returned to the humidified CO₂ incubator (32° C.,10% CO₂) for an additional seven days, after which the wells weresubjected to freezing at −20° C. overnight followed by thawing andresuspension by repeated pipetting. A 100 μl aliquot of thefreeze/thawed transfected cells was transferred to each well of a platewith fresh PER.C6/E2A cells seeded on normal 96-well-tissue cultureplates (plate 2) as described above. The second 96-well plate, withPER.C6/E2A cells incubated and thus infected with freeze/thawed celllysate of the first transfected plate, was checked for CPE formation(see FIG. 27) and stored at −20° C. In FIG. 27, the percentage of virusproducing cells (CPE positive wells) scored after propagation of thefreeze/thawed transfected cells to new PER.C6/E2A cells is depicted.Clearly, the miniaturized system subject of this application allows theefficient production of deltaE1/E2A double deleted vectors with avariety of transgene inserts.

Example 11 Quantification of Adenoviral Vector Particles Produced inMiniaturized Production System Using PER.C6/E2A

Adenoviral plaque assays were performed in order to determine the titerof the adenoviral vectors produced in one well of a96-well-tissue-culture plate. PER.C6/E2A cells were harvested usingtrypsin-EDTA and counted. The cells were then diluted with culturemedium (DMEM with 10% fetal bovine serum and 10 mM MgCl₂) to asuspension of 1.5×10⁶ cells per 2 ml, followed by seeding 2 ml per wellon PLL coated 6-well plates (Becton Dickinson). Microtiter platescontaining adenoviral vector lysates were thawed and 50 μl of a randomlychosen well of each adenovirus was used to make serial 10-fold dilutionsof the adenovirus in culture medium. The medium of the PER.C6/E2A cells,which were seeded the same day, was replaced with 2 ml per well dilutedvirus and the 50–60% monolayer was infected for approximately 16 hoursin a humidified CO₂ incubator (32° C., 10% CO₂). After infection, thecells were overlayed with 3 ml per well agarose mix (2×MEM, 2% fetalbovine serum, 1 mM MgCl₂, PBS, and 1% agarose) and returned to thehumidified CO₂ incubator (32° C., 10% CO₂). After two weeks, nineindividual plaques, including one negative control, were transferred to200 μl of culture medium and stored at −20° C. An aliquot of 25 μl ofthis material was used to infect PER.C6/E2A cells (2.25×10⁴ cells perwell in 100 μl), seeded in 96-well tissue culture plates one day priorto infections. This was incubated in the humidified CO₂ incubator (32°C., 10% CO₂) until the presence of full CPE was observed and wassubsequently stored at −20° C.

The final titer of the adenoviruses, produced in a well of a 96-welltissue culture plate, was determined one week after picking theindividual plaques. In FIG. 28, the titer of adenoviruses, in pfu/ml,produced in a well of a 96-well plate is depicted. Average titers of0.8±0.7×10⁹ pfu/ml imply that depending on the MOI needed in aparticular cell based assay in a functional genomics screen using384-well plates, sufficient virus is produced for 400–4000 assays (MOIsof 100–10). This allows multiple screens using one library.

Example 12 The Quality of Adenoviral Vector Produced in a MicrotiterPlate on PER.C6/E2A Cells

To test for functionality of the produced recombinant adenovirus, thefollowing functional assays were performed on cells infected with therespective adenoviral vectors: β-Galactosidase assay, hIL3 assay,luciferase assay, ceNOS assay, GLVR2 assay, and EGFP assay.

β-Galactosidase Assay

A549 cells were harvested using trypsin-EDTA and counted. The cells werethen diluted with culture medium (DMEM with 10% heat-inactivated FBS) toa suspension of 10,000 cells per 100 μl, followed by seeding 100 μl perwell of 96-well tissue culture plates. The next day, all CPE-positivePER.C6/E2A wells containing lacZ-transducing adenoviruses, as well asnegative controls (both primary wells and plaques amplified on freshPER.C6/E2A cells), were used to infect the A549 cells. For this purpose,the frozen wells were thawed and 20 μl of each well of the freeze/thawedcell lysate was used to infect one well of the A549 cells. Two daysafter infection, the medium of the infected A549 cells was removed andeach well was washed two times with 100 μl PBS (phosphate-bufferedsaline). After washing, the cells were fixated for five minutes at roomtemperature by adding 100 μl fixative (1% formaldehyde, 0.2%glutardialdehyde) per well. After washing the cells two times with PBS,100 μl X-gal staining solution (0.2 M K₃Fe(CN)₆, 0.2 M K4Fe(CN)₆, X-galin DMSO and 0.1 M MgCl₂) was added to each well.

All of the wells that were infected with CPE-positive wells stainedblue. A large number of wells showed 100% blue staining implyingtransduction of all cells with adenoviral vector carrying lacZ (see FIG.29).

hIL-3 Assay

The day before infection, the A549 cells were seeded as described above.The next day, all CPE-positive PER.C6/E2A wells containing humaninterleukin-3 (hIL-3) transducing adenoviruses (both primary wells andplaques amplified on fresh PER.C6/E2A cells), as well as positive andnegative controls, were used to infect the A549 cells. For this purpose,the frozen wells were thawed and 20 μl of each well of the freeze/thawedcell lysate was used to infect one well of the A549 cells. Three daysafter infection, the quantity of hIL-3 concentrations in 100 μl of thesupernatants of the infected A549 cells was determined using the humanIL-3 immunoassay (Quantikine™).

All of the wells that were infected with CPE-positive wells showed highhIL-3 concentrations (see FIG. 29).

Luciferase Assay

The day before infection, the A549 cells were seeded as described above.The next day, all CPE-positive PER.C6/E2A wells containing luciferasetransducing adenoviruses (both primary wells and plaques amplified onfresh PER.C6E2A cells), as well as positive and negative controls, wereused to infect the A549 cells. For this purpose, the frozen wells werethawed and 20 μl of each well of the freeze/thawed cell lysate was usedto infect one well of the A549 cells. Two days after infection, themedium of the infected A549 cells was removed and each well was washedwith 100 μl PBS. After adding 100 μl 1× reporter lysis buffer (Promega),the wells were subjected to freeze/thawing followed by measuring theluciferase activity in 20 μl of the freeze/thawed cell lysates.

All of the wells that were infected with CPE-positive wells showed ahigh luciferase activity (see FIG. 29).

ceNOS Assay

PER.C6/E2A cells were harvested using trypsin-EDTA and counted. Thecells were then diluted with culture medium (DMEM devoid of phenol-redwith 10% FBS and 10 mM MgCl₂) to a suspension of 22,500 cells per 100μl, followed by seeding 100 μl per well of 96-well tissue cultureplates. The next day, all CPE-positive PER.C6/E2A wells containing ceNOStransducing adenoviruses (both primary wells and plaques amplified onfresh PER.C6/E2A cells), as well as positive and negative controls, wereused to infect the PER.C6/E2A cells. For this purpose, the frozen wellswere thawed and 20 μl of each well of the freeze/thawed cell lysate wasused to infect one well of the PER.C6/E2A cells. Three days afterinfection, 50 μl color solution [GreissA reagent (0.1%N-(1-Naphthyl)Ethylenediamine) and GreissB reagent (25% Sulfanylamide in5% phosphoric acid) in a 1:1 ratio] was added to 50 μl of thesupernatants of the infected PER.C6E2A cells. After adding the colorsolution, supernatants with a positive ceNOS activity turned pink.

All of the wells that were infected with CPE-positive wells showed apositive ceNOS activity (see FIG. 29).

GLVR2 Amphotropic Receptor Assay

Adenoviral mediated transduction of GLVR2, the receptor for amphotropicretroviruses, was measured essentially as described (Lieber et al,1995), except for the use of an amphotropic retroviral supernatanttransferring a truncated version of the human nerve growth receptor(NGFR). Retroviral transduction of the CHO cells infected with GLVR2adenoviral supernatant was detected using anti-NGFR antibodies and aflow cytometer.

All of the wells that were infected with CPE-positive PER.C6/E2A wellscontaining GLVR2 transducing adenoviruses (plaques amplified on freshPER.C6/E2A cells) showed a positive GLVR2 activity (see FIG. 29).

EGFP Assay

EGFP expression was measured on a microtiter plate fluorimeter or byflow cytometer.

In conclusion, virus produced from wells as well as virus plaquepurified (i.e., cloned) from producing wells showed transduction oftheir respective transgenes. Therefore, the system shows high fidelityfor the production of functional adenoviral vectors and produces noaberrant forms for the transgene inserts tested.

Example 13 DNA Isolation Methods Generating Sufficiently PurifiedPlasmid DNA for Production of Adenoviral Vectors in PER.C6 andPER.C6-E2A Cells

It is well known that plasmid DNA that is used for transfection studiesin eukaryotic cells must be of sufficient purity and free of endotoxinsto achieve high levels of transfection efficiencies. Conventionalmethods for purifying plasmid DNA from E. coli include an alkaline lysisprocedure (Birnboim, H. C. and Doly, J, (1979) and a rapid alkalinelysis procedure for screening recombinant plasmid DNA (Nucleic Acid Res.7: 1513–1522) followed by either banding of the plasmid DNA on cesiumchloride (CsCI) gradients (see Sambrook, J. et al, eds. (1989) Molecularcloning: a laboratory manual, 2^(nd) edition, Cold Spring HarborLaboratory Press) or by binding and elution on an anion-exchange resin(see, for example, Qiagen™ plasmid purification methods of Qiagen Inc.;and Concert™ plasmid purification systems of Life Technologies).However, all of these methods are unsuited for high throughput DNAisolations because they require considerable hands-on time perisolation. Therefore, to reduce the amount of time and costs perisolation, other methods were examined.

Methods that were examined using the SalI linearized adenoviral adapterplasmid pCLIP-SalI LacZ and the E2A deleted helper fragmentpWE/Ad.AflII-rITR.deltaE2A: alkaline lysis followed by column basedplasmid purification (Qiagen)

-   -   1. alkaline lysis followed by isopropanol precipitation, and        solubilization in TE buffer    -   2. alkaline lysis followed by isopropanol precipitation, and        solubilization in TE buffer containing RNAse at 10 microgram per        ml    -   3. alkaline lysis followed by isopropanol precipitation, and        solubilization in TE buffer containing RNAse at 10 microgram per        ml, followed by phenol/chloroform extraction and ethanol        precipitation    -   4. Standard cetyltrimethylammonium bromide (CTAB) plasmid        isolation (Nucleic Acids Res, 16²⁰;1488)    -   5. Standard CTAB plasmid isolation, but solubilization in TE        buffer containing RNAse at 10 microgram per ml, followed by        phenol/chloroform extraction

Equal volumes of the resulting plasmids were linearized with SalI,followed by phenol/chloroform extraction and ethanol precipitation.Following solubilization in TE buffer and verification on an agarosegel, equal amounts of DNA (as determined by the ethidium bromidestaining) were transfected into PER.C6/E2A cells with lipofectamine asdescribed under examples 9 and 10. After propagation, wells were scoredfor CPE formation as a measure of virus production.

In FIG. 30, the percentage of wells showing CPE formation (CPE positive)after transfection of PER.C6/E2A cells transfected with pCLIP-LacZ,purified by 6 different protocols, is represented. Qiagen: standardalkaline lysis followed by Qiagen plasmid purification; AlkLys: alkalinelysis followed by isopropanol precipitation and solubilization in TEbuffer; AL+RNAse: alkaline lysis followed by isopropanol precipitationand solubilization in TE buffer containing RNAse at 10 microgram per ml;AL+R+phenol: alkaline lysis followed by isopropanol precipitation andsolubilization in TE buffer containing RNAse at 10 microgram per ml,followed by phenol/chloroform extraction and ethanol precipitation;CTAB: standard CTAB plasmid isolation; CTAB+phenol: standard CTABplasmid isolation, but solubilization in TE buffer containing RNAse at10 microgram per ml is followed by phenol/chloroform extraction. It isevident that the quality of DNA is not a major determinant fortransfection of, and virus production in, PER.C6/E2A cells, as all 6differently isolated plasmids produced similar numbers of wells withCPE.

In conclusion, for high throughput transfection of, and virus productionin, PER.C6/E2A cells, it is sufficient to use plasmid DNA that wasprecipitated with 0.6 volumes of isopropanol after standard alkalinelysis, followed by solubilization in TE buffer.

Example 14 The Use of Unpurified, Digested Adapter and Helper AdenoviralDNA Molecules for the Generation of Adenoviral Vectors in a MiniaturizedFormat

In order to minimize the overall costs and chances for errors in theprocedure, and to maximize the throughput when producing recombinantadenoviruses in a high throughput fashion, it is desirable to eliminateas many steps as possible. Any improvement here is also applicable whengenerating adenoviral vectors on a smaller, low throughput scale. Themost difficult step to automate when producing recombinant adenovirusesis the DNA clean-up step by phenol/chloroform extraction (p/c) prior totransfection of cells with the DNA. DNA is purified after linearizationin order to obtain enyzme-free DNA. This is thought to be important toobtain high percentages of viral generation after transfection with theadapter and helper DNA molecules. An additional motivation to eliminatethe p/c purification procedure is the risk of traces of phenol andchloroform in the DNA used for transfection, which can have a negativeeffect on the generation of viruses. Therefore, it was investigatedwhether the complicated p/c purification step could be omitted from theminiaturized adenoviral vector generation protocol subject of thisapplication. This method forms the basis of high throughput constructionof libraries, such as sense or antisense cDNA expression libraries.Several independant experiments were performed in order to test theeffect of omitting the p/c step on the efficiency of adenoviral vectorgeneration. The p/c purification was carried out as follows: afterdigesting the adapter-DNA and rITR-DNA with the appropriate restrictionenzymes, an equal volume of phenol and chloroform (1:1) was added, mixedthoroughly, and centrifuged (5 minutes, 14,000 rpm). The aqueous phasewas transferred to a new micro-centrifuge tube and an equal volume ofchloroform was added. Again, this was mixed thoroughly and centrifuged(5 minutes, 14,000 rpm). The aqueous phase was transferred to a newmicrocentrifuge tube and 0.1 times the volume of 3 M sodium acetate (pH5.2) and 2.5 times the volume of absolute ethanol were added. Themixture was kept at −20° C. for at least 20 minutes, subsequentlycentrifuged (15 minutes, 14,000 rpm), and the pellet was washed with 70%ethanol. The DNA was air-dried and a suitable volume of sterile waterwas added (in Laminar Airflow Cabinet). Transfection was carried out asdescribed in examples 9 and beyond using PER.C6/E2A cells. All viruseswere E1 and E2A deleted and were produced in PER.C6/E2A cells.

In the first experiment, adapter-DNA containing β-galactosidase(pAd5.Clipsal.LacZ) of 6 different DNA isolation protocols (as describedin example 13) were analyzed and compared for their efficiency inproducing adenoviral vector by monitoring for CPE formation. Half of theDNA samples were p/c purified after linearization using the appropriaterestriction enzyme (SalI) while the remaining half of the samples werenot purified after linearization. The restriction enzyme was heatinactivated to exclude inadvertant digestion of the helper DNA because aSalI site is present in the rITR delta E2A helper fragment. In thisexperiment, p/c purified rITR delta E2A helper DNA was used. In all ofthe DNA isolation methods, CPE was formed efficiently (FIG. 31A). Insome cases, elimination of the p/c purification step gave higher CPEefficiencies. In conclusion, adenoviral adapter DNA digested with thelinearizing enzyme can be used for transfection without priorpurification.

In the second experiment, the difference between using a purificationstep and not using a purification step were compared using adapter-DNAcontaining Enhanced Green Fluorescent Protein (EGFP) and Enhanced YellowFluorescent Protein (EYFP) (pAd5.Clippac.EGFP and pAd5.Clippac.EYFP).The adapter plasmid-DNA was isolated using the Qiagen isolation method.The rITR delta E2A used was p/c purified. The adapter-DNA was linearizedusing PacI, which did not have to be heat inactivated beforetransfection because there is no PacI site in the rITR. No consistentdifferences were found in the percentages of CPE observed and productionof adenoviral vector was efficient (FIG. 31B).

The third experiment tested the need to purify the rITR. The adapter-DNAcontained EGFP (pAd5.Clippac.EGFP) and was isolated using the Qiagenisolation method (FIG. 30C). The results after transfection andpropagation show that the purification of both adapter and rITR DNAafter digestion is not necessary.

Taking all results in account, it is clear that the phenol chloroformpurification step is not necessary to obtain high percentages of CPE andfor adenoviral vector production. The above described modification ofthe procedure, as for example described in examples 9 and 10, results ina significant increase in throughput when generating adenoviral vectorlibraries in an automated setup and when making vectors manually on asmaller scale.

Example 15 Production of Adenoviral Vectors in Relation to Stability ofthe Produced Vector

Generation of recombinant denoviruses, as described in the variousexamples herein, indicates that a functional adenovirus will be formedapproximate five to eleven days after amplification of the virusproduced on the transfected PER.C6 cells, and their derivatives, grownin multiwell tissue culture plates. The observation of a cytopathiceffect (CPE) indicates that functional adenovirus has been formed and isreplicating. The nature of the transgene inserts and variations in theexperimental conditions cause the kinetics of virus generation to bevariable. In a high throughput setting where large numbers of wells andplates containing adenoviral vector are handled, it is desirable to havea single point in time to harvest the plates and score for adenoviralvector production. The above mentioned variations in adenoviral vectorgeneration may be overcome by postponing the harvest of the plates aslong as possible, i.e. until the slower wells also have producedadenoviral vector. For this purpose, the stability of recombinant lacZadenovirus (pCLIP-lacZ), once it is produced starting from low numbersof virus to higher numbers of virus, was tested. Then, the titers (seeexample 11) and lacZ transduction-potential of the virus after up tothree weeks were determined.

PER.C6/E2A cells were seeded in two rows of 96-well microtiter tissueculture plates using 4×10⁴ cells/well. The plates were incubatedovernight at 39° C. The next day, PER.C6/E2A was infected with purifiedLacZ-adenoviral vector of serotype 5. The infections were done atdifferent MOIs according to the scheme below (21 plates in total).

TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 A B 0.01 0.01 0.01 0.1 0.1 0.1 1 1 110 10 10 C 0 0 0 0 0 0 0 0 0 0 0 0

To determine the effect of temperature on stability of adenovirusesproduced in wells, seven plates were incubated at 32° C., seven platesat 34° C., and seven plates at 39° C. At days 2, 3, 6, 9, 13, 16, and 21after infection, one plate corresponding to each incubation temperaturewas frozen. The cell lysates were used in the following experiments.

In order to determine the transduction potential of the producedadenoviruses, A549 cells were seeded in 96-well microtiter tissueculture plates with 1×10⁴ cells/well and incubated overnight at 37° C.Then, the cells were infected with 50 μl cell lysate and incubated at37° C. After two days, the cells were screened for toxicity followed bylacZ staining. A clear toxic effect was observed with increasing MOI andincreasing time of infection. The table below is a summary of when allcells stained blue in all wells.

TABLE 2 # days after moi infection. 32° C. 0.001 9 0.01 9 0.1 6 1 6 34°C. 0.001 6 0.01 6 0.1 6 1 6 37° C. 0.001 6 0.01 6 0.1 3 1 3

Three weeks after infection, 100% of blue cells/well were still observedand thus all cells with adenoviral vector carrying lacZ. Thus, thisshowed no decreased infectivity upon incubation up to 3 weeks.

In order to determine the number of infectious viral particles in thecell lysate, a titration assay was performed for the samples which wereincubated 2, 6, and 21 days after infection corresponding to eachincubation temperature and MOI. Three weeks after infection, an averagetiter of 2×10¹⁰ pfus per ml was observed. An overview of the titers isgiven in FIG. 32.

The above mentioned experiments indicate that variations in adenoviralvector generation may be overcome by postponing the harvest of theplates as long as possible, i.e. until the slower wells also haveproduced adenoviral vector. Although a clear toxic effect is seen withincreasing MOI and increasing time of infection, there is no decreasedinfectivity and no decrease in titer of the produced adenoviral vector.

Example 16 Miniaturized Production of Adenoviral Vectors CarryingAntisense DNA Sequences and Expressing Antisense mRNA Sequences

Decreasing endogenous gene expression in screens using antisense cDNAexpression libraries is very useful in functional genomics programs.Individual antisense adenoviral vectors can also be used for genevalidation and the devlopment of an antisense gene therapeutic. Anexample is the use of antisense-Vascular Endothelial Growth Factor(VEGF). VEGF is a pivotal molecule in tumoral angiogenesis that promotesendothelial cell growth and plays a major role in neovascularization andgrowth of gliomas. The VEGF-antisense molecule inhibits tumor growth invivo. (Seock-Ah et al., 1999, Cancer research 59, 895–900). Constructinglarge, complex antisense libraries in adenoviral vectors are valuableand very useful for Functional Genomics screening programs.

PER.C6/E2A cells were cotransfected with linearized adapter DNA,containing a defined human cDNA sequence in antisense orientation, andlinearized rITR delta E2A helper DNA, as described in example 10. Thegenes cloned in antisense orientation in adapter DNA are described inTable 3. For pCLIP, two variants were used with SalI or PacI as the siteto linearize depending on the transgene inserts.

TABLE 3 Antisense-cDNA Abbreviation Adenoviral vector constitutivenitric oxide CeNOS pClIP, pIPspAdapt synthase lysosomal beta- hGC pCLIPglucocerebrosidase Phenol UDP- P-UGT pCLIP, pIPspAdaptglucuronosyltransferase Bilirubin 1 UDP- B-UGT pCLIP, pIPspAdaptglucuronosyltransferase Plasminogen Activator PAI-1 pCLIP, pIPspAdaptInhibitor type-1 Ribosomal protein L4 NA pCLIP Phosphoenolpyruvate PEPCKpCLIP carboxykinase β-globin NA pCLIP Lysozyme NA pCLIP Chrom 1 spec.transcr. KIAA pCLIP KIAA0493 snRNP core protein Sm D2 SnRNP pCLIP

In FIG. 33, an example is given of some of the above mentioned cDNAs forgeneration of antisense cDNA adenoviral vectors using the miniaturizedproduction system subject of this invention. These viruses will be usedto attempt to lower the endogenous expression of the cells tested.

Example 17 Construction of Adapter Plasmids for the Generation andProduction of Recombinant Adenoviruses, in Particular, for theGeneration and Production of Adenoviral Expression Libraries

Adenoviral adapter plasmids (“adapter”) were constructed that containmultiple cloning sites in multiple orientations that allow efficientcloning of sense or anti-sense cDNA sequences and the generation oflibraries of nucleic acids including cDNA libraries in these vectors.Furthermore, these new adapter plasmids contain novel restriction enzymerecognition sequences bordering the left adenoviral ITR and thesequences overlapping with the helper fragment up to nucleotide 6095 ofthe Ad5 viral genome. These modifications of the adenoviral adapterplasmids significantly enhance the possibility to linearize the adapterplasmids, without digestions of inserted transgenes or transgenelibraries. Following cotransfection with pWE/Ad.AflII-rITR.deltaE2A,homologous recombination between the improved adapter plasmids and theadenoviral cosmid results in the generation of functional adenoviruses.

The first adapter constructs, pCLIP-IppoI (FIG. 34A) andpCLIP-IppoI-polynew (FIG. 34B), are derived from pAd5/ CLIP-Pac andcontain a new I-PpoI linearization site at position −11 bp in front ofthe left ITR. In addition, pCLIP-IppoI-polynew contains an improvedpoly-linker sequence downstream of the CMV promoter encompassingrestriction enzyme recognition sequences for different, rare-cuttingrestriction endonucleases and intron-encoded endonucleases. Therecognition sequences for intron-encoded endonucleases are extremelyrare in genomes, including the human genome, and consist of 11–23 basepairs. As these intron-encoded endonuclease sites are absent in theadenoviral genome, sequences can be directly inserted into a fulladenoviral vector genome obtained from an insertless pCLIP-IPpoI (seealso below).

To construct this adapter plasmid, part of the left ITR of Ad5 wasamplified by PCR on pCLIP-PacI template plasmid DNA using the followingprimers: PCLIPPACIPPO: 5′-TTT TTA ATT AAT AAC TAT GAC TCT CTT AAG GTAGCC AAA TCA TCA TCA ATA ATA TAC CTT ATT TTG G-3′ (SEQ ID NO:48) andPCLIPBSRGI: 5′-GCG AAA ATT GTC ACT TCC TGT G-3′ (SEQ ID NO:49) andElongase polymerase from Life Technologies (LTI; Breda, NL). PrimerpCLIP-PacI contains a PacI site 5′ from a I-PpoI sequence. The amplifiedfragment was digested with PacI and BsrGI and the resulting 255 bpfragment cloned into a fragment of 6471 bp, which was obtained frompAd5/CLIP-PacI digested with the same enzymes and isolated on a 1%agarose gel. Nucleotide sequences were confirmed by dideoxynucleotidesequence analysis. This construct, containing PacI and I-PpoIrecognition sequences 5′ to the left ITR at a distance of 33 nucleotidesand 11 nucleotides, respectively, was named pCLIP-I-PpoI (see FIG. 34A).This construct was subsequently digested with XbaI and HindIII,separated on a gel, and used to insert a new synthetic linker sequence.This linker sequence, composed of the two single stranded and thefollowing annealed oligonucleotides LINKERPOLYNEW-S: 5′-AGC TTT AAC TATAAC GGT CCT AAG GTA GCG ATT AAT TAA CAG TTT AAT TAA TGG CAA ACA GCT ATTATG GGT ATT ATG GGT T-3′ (SEQ ID NO:50); and LINKERPOLYNEW-AS: 5′-CTAGAA CCC ATA ATA CCC ATA ATA GCT GTT TGC CAT TAA TTA AAC TGT TAA TTA ATCGCT ACC TTA GGA CCG TTA TAG TTA A-3′ (SEQ ID NO:51), was directlyligated into the digested construct. This adapter construct, termedpCLIP-I-PpoI-polynew, now contains recognition sequences for therestriction enzymes HindIII, I-CeuI, PacI, Pi-PspI, and XbaI in thepolylinker (see FIG. 34B). Correct insertion of this linker was verifiedby digestions with the respective enzymes and sequence analysis.

A different adapter construct, pADAPT, which contains a stronger CMVpromoter than pCLIP-based adenoviral adapters, as well as a differentpoly(A) sequence, was used as a backbone to construct another set ofadapter plasmids. To enhance the linearization possibilities, a numberof pADAPT derivatives were designed and constructed. For this purpose,pADAPT plasmid DNA was digested with SalI and treated with ShrimpAlkaline Phosphatase to reduce religation. A linker, composed of thefollowing two phosphorylated and annealed oligonucleotides ExSalPacF5′-TCG ATG GCA AAC AGC TAT TAT GGG TAT TAT GGG TTC GAA TTA ATT AA-3′(SEQ ID NO:52) and ExSalPacR 5′-TCG ATT AAT TAA TTC GAA CCC ATA ATA CCCATA ATA GCT GTT TGC CA-3′ (SEQ ID NO:53), was directly ligated into thedigested construct, thereby replacing the SalI restriction site withPi-PspI, SwaI, and PacI. Furthermore, part of the left ITR of pADAPT wasamplified by PCR using the following primers: PCLIPMSF: 5′-CCC CAA TTGGTC GAC CAT CAT CAA TAA TAT ACC TTA TTT TGG -3′ (SEQ ID NO:54) andpCLIPBSRGI (see above). The amplified fragment was digested with MunIand BsrGI and cloned into pCLIP-EcoRI, which was partially digested withEcoRI and after purification digested with BsrGI. After restrictionenzyme analysis, the construct was digested with ScaI and SgrAI and an800 bp fragment was isolated from an agarose gel and ligated into aScaI/SgrAI digested pADAPT+ExSalPac linker. The resulting construct,named pIPspSalAdapt (see FIG. 34C), was digested with SalI,dephosphorylated, and ligated to the above-mentioned phosphorylatedExSalPacF/ExSalPacR doublestranded linker. A clone in which PacI sitewas closest to the ITR was identified by restriction analysis andsequences were confirmed by sequence analysis. This novel pADAPTconstruct, termed pIPspAdapt (see FIG. 34D), thus harbours two ExSalPaclinkers containing recognition sequences for PacI, PI-PspI, and BstBI,which surround the adenoviral part of the adenoviral adapter constructand can be used to linearize the plasmid DNA prior to cotransfectionwith adenoviral helper fragments.

In order to further increase transgene cloning permutations, a number ofpolylinker variants were constructed based on pIPspAdapt. For thispurpose, pIPspAdapt was first digested with EcoRI and dephosphorylated.A linker composed of the following two phosphorylated and annealedoligonucleotides: Ecolinker⁺: 5′-AAT TCG GCG CGC CGT CGA CGA TAT CGA TAGCGG CCG C 3′ (SEQ ID NO:55) and Ecolinker⁻: 5′-AAT TGC GGC CGC TAT CGATAT CGT CGA CGG CGC GCC G 3′ (SEQ ID NO:56) was ligated into thisconstruct, thereby creating restriction sites for AscI, SalI, EcoRV,ClaI and NotI. Both orientations of this linker were obtained andsequences were confirmed by restriction analysis and sequence analysis.The plasmid containing the polylinker in the order 5′ HindIII, KpnI,AgeI, EcoRI, AscI, SalI, EcoRV, ClaI, NotI, NheI, HpaI, BamHI, and XbaIwas termed pIPspAdapt1 (see FIG. 34E) while the plasmid containing thepolylinker in the order HindIII, KpnI, AgeI, NotI, ClaI, EcoRV, SalI,AscI, EcoRI, NheI, HpaI, BamHI and XbaI was termed pIPspAdapt2 (see FIG.34F).

Those skilled in the art of making cDNA libraries will appreciate thatan extra polylinker, consisting of the oligonucleotides GalMlu-F: 5′-CGATCG GAC CGA CGC GTT CGC GAG C-3′ (SEQ ID NO:57) and GalMlu-R: 5′-GGC CGCTCG CGA ACG CGT CGG TCC GAT-3′ (SEQ ID NO:58), was inserted in betweenthe ClaI and NotI sites of pIPspAdapt1, to generate pIPspAdapt6 (seeFIG. 34G). pIPspAdapt6 contains extra restriction sites for RsrII, MluI,and NruI, which were introduced to increase the distance between theSalI and NotI sites in order to improve the digestion of the combinationof these enzymes. Furthermore, the enzymes allow the pre-digestion anddephosphorylation of this vector prior to restriction with SalI andNotI, which will reduce background recombinants in the case of cloningindividual inserts or libraries with SalI- and NotI-compatibleoverhangs. The GalMlu oligo was also cloned into pIPspAdapt2, leading topIPspAdapt7 (see FIG. 34H).

To facilitate the cloning of additional sense or antisense constructs, alinker composed of the following two oligonucleotides was designed toreverse the polylinker of pIPspAdapt: HindXba⁺ 5′-AGC TCT AGA GGA TCCGTT AAC GCT AGC GAA TTC ACC GGT ACC AAG CTT A-3′ (SEQ ID NO:59);HindXba⁻ 5′-CTA GTA AGC TTG GTA CCG GTG AAT TCG CTA GCG TTA ACG GAT CCTCTA G-3′ (SEQ ID NO:60). This linker was ligated into HindIII/XbaIdigested pIPspAdapt and the correct construct was isolated. Confirmationof the correct construct was done by restriction enzyme analysis andsequencing. This new construct, pIPspAdaptA (see FIG. 34I), was digestedwith EcoRI and the above mentioned Ecolinker was ligated into thisconstruct. Both orientations of this linker were obtained, resulting inpIPspAdapt3 (see FIG. 34J), which contains the polylinker in the orderXbaI, BamHI, HpaI, NheI, EcoRI, AscI, SalI, EcoRV, ClaI, NotI, AgeI,KpnI, and HindIII, and pIPspAdapt4, which contains the polylinker in theorder XbaI, BamHI, HpaI, NheI, NotI, ClaI, EcoRV, SalI, AscI, EcoRI,AgeI, KpnI and HindIII (see FIG. 34K). All sequences were confirmed byrestriction enzyme analysis and sequencing.

As mentioned above, intron-encoded endonucleases are rare, cuttingenzymes and do not digest the adenoviral genome. Those skilled in theart will appreciate that these enzymes allow the direct ligation ofsequences in the adenoviral genome, since they do not have a recognitionsequence in the adenoviral genome. To obtain a pADAPT version thatcontains recognition sequences for intron-encoded endonucleases in thepolylinker, a linker was ligated into HindIII/XbaI digested pIPspAdapt,consisting of the single stranded sequences: 5′-AGC TTA ACT ATA ACG GTCCTA AGG TAG CGA TAG GGA TAA CAG GGT AAT TAA TTA ATT TAA ATT AAT TAA TCTATG TCG GGT GCG GAG AAA GAG GTA ACT ATG ACT CTC TTA AGG TAG CCA AAT-3′(SEQ ID NO:61); and 5′-CTA GAT TTG GCT ACC TTA AGA GAG TCA TAG TTA CCTCTT TCT CCG CAC CCG ACA TAG ATT AAT TAA TTT AAA TTA ATT AAT TAC CCT GTTATC CCT ATC GCT ACC TTA GGA CCG TTA TAG TTA-3′ (SEQ ID NO:62). Thislinker was composed of four oligonucleotides (IntrolinkerF1,IntrolinkerF2, IntrolinkerR1, and IntrolinkerR2) and containsrecognition sequences for the intron-encoded endonucleases I-CeuI,I-SceI, PI-SceI, and I-PpoI and the endonucleases PacI and SwaI. Thecorrectness of the construct, termed pIPspAdapt5 (see FIG. 34L), wasconfirmed by sequence analysis.

Adenoviral DNA from viruses containing pIPspAdapt5 or pCLIPIppoI-polynewwas isolated and cloned into the cosmid vector pWE15/SnaB1. pWE15/SnaB1was created by auto-annealing the phosphorylated oligonucleotide PacSna:5′-TAA TAC GTA TTA AT-3′ (SEQ ID NO:63) and ligating the resultingdouble stranded sequence in PacI-digested and dephosphorylatedpWE15/PAC, a derivative of pWE15 (see Sambrook, J. et al, eds. (1989)Molecular cloning: a laboratory manual, 2^(nd) edition, Cold SpringHarbor Laboratory Press). This generates a restriction site for SnaBl,which is flanked by PacI sites. For the generation of adenoviralDNA-containing cosmid, blunt-ended adenoviral DNA was isolated accordingto standard laboratory procedures using Dnase and Proteinase K, followedby elution on an anion-exchange resin spin column. A molar excess of theresulting purified adenoviral DNA was ligated into SnaB1-restrictedpWE15/SnaB1 and the resulting ligation mixture was transfected into E.coli Stb12 cells (LTI, Breda).

The resulting plasmid DNA was subsequently used for in vitro ligations(see FIG. 34M). The use of pIPspAdapt5-derived cosmid DNA will be usedan an example in the following: double stranded oligonucleotidescontaining compatible overhangs were ligated between the I-CeuI andPI-SceI sites, between I-CeuI and I-PpoI, between I-SceI and PI-SceI,and between I-SceI and I-Ppol. The PacI restriction endonuclease wassubsequently used not only to linearize the construct after ligation andthereby to liberate the left- and right ITRs, but also to eliminatenon-recombinants. In this case, ligation mixtures can be used directlyfor transfection in PER.C6/PER.C6/E2A packaging cells or variantsthereof, thereby eliminating the need for a cross-over or homologousrecombination event to generate functional adenovirus.

As an alternative, adapter plasmids and cosmids containing adenoviralDNA, which was made from pIPspAdapt5 or pCLIP-IppoI-polynew, were usedto generate fragments either encompassing the region between the leftITR and the first part of the polylinker or encompassing the second partof the polylinker until the right ITR. Care is taken that the left andright ITR are linearized with distinct and non-compatible restrictionenzymes since ligation efficiencies are otherwise strongly reduced.pIPspAdapt5-derived cosmid DNA will be used as an example: plasmidpIPspAdapt5 was cut with either BstBI and I-CeuI or BstBI and I-SceI togenerate the adenoviral fragment containing the left ITR. The cosmidcontaining the pIPspAdapt5-derived adenoviral DNA was restricted withI-PpoI and PacI or PI-SceI and PacI to generate the fragment containingthe right ITR. Fragments containing the left and right ITR were isolatedon a 0.8% agarose gel and purified using anion exchange resins.Subsequently, double stranded oligonucleotides containing compatibleoverhangs for either I-CeuI or I-SceI at the 5′ end and I-PpoI orPI-SceI at the 3′ end were ligated in equimolar amounts with thefragments containing the left and right ITRs. The resulting ligationmixture was used for transfection into PER.C6/PER.C6/E2A packaging cellsor variants thereof, again eliminating the need for a cross-over orhomologous recombination event to generate functional adenovirus.

The direct transfection of in vitro ligated products benefits from analternative method of isolating the adenoviral vector DNA. To improvethe efficiency of viral production after transfection of in vitroligation reactions, adenoviral vector DNA can be isolated from purifiedadenoviral particles (see Pronk, R. et al., Chromosoma 102: S39–S45(1992)). This virion DNA contains two molecules of Terminal Protein (TP)covalently bound to the ITR sequences. It is known that TP-DNAstimulates adenoviral DNA replication over 20 fold compared toprotein-free DNA.

Therefore, pIPspAdapt5- or pCLIP-IppoI-polynew-derived adenoviral DNAcan be isolated from virions using guanidinium hydrochloride asdescribed (Van Bergen, B. et al. Nucleic Acids Res. 11: 1975–1989(1983)). This DNA was digested with a suitable combination ofintron-encoded restriction endonucleases and used for in vitro ligationreactions. After ligation, non-recombinants were removed by digestionwith PacI. Further procedures were as described above and in example 10and beyond. pIPspAdapt adapter plasmids were co-transfected withpWE/Ad.AflII-rITRDE2A in the PER.C6/E2A packaging cells to generaterecombinant adenoviruses, as is shown in FIG. 34N using pIPspAdapt2 asan example.

Example 18 E1-deleted or E1+E2A-deleted Recombinant Adenoviruses withDeletions in the E3 Region for Cloning of Larger DNA Inserts inMiniaturized Adenoviral Vector Production System

It is known that none of the E3-encoded proteins is required foradenoviral replication, packaging, and infection in cultured cells. Thisallows the possible removal of the E3 region from recombinantadenoviruses, creating the opportunity for inserting large genes orcomplex regulatory elements without exceeding the maximal packagingcapacity. For example, part of the E3 region can be removed by deletinga XbaI-XbaI fragment, corresponding to Ad5 wt sequence 28592–30470.Another example is an expanded deletion of the E3 region in whichsequences between the stop codon of pVIII and the translation initiationcodon of fiber, corresponding to Ad5 wt sequence 27865–30995, wereremoved.

Generation of pWE/Ad.AflII-rITRΔE2A

Deletion of the E2A coding sequences from pWE/Ad.AflII-rITR (ECACCdeposit P97082116) has been accomplished as follows: the adenoviralsequences flanking the E2A coding region at the left and the right siteswere amplified from the plasmid pBr/Ad.Sal.rITR (ECACC depositP97082119) in a PCR reaction with the Expand PCR system (Boehringer)according to the manufacturer's protocol. The following primers wereused:

Right flanking sequences (corresponding Ad5 nucleotides 24033 to 25180):ΔE2A.SnaBI: 5′-GGC GTA CGT AGC CCT GTC GAA AG-3′ (SEQ ID NO:64)ΔE2A.DBP-start: 5′-CCA ATG CAT TCG AAG TAC TTC CTT CTC CTA TAG GC-3′(SEQ ID NO:65) The amplified DNA fragment was digested with SnaBI andNsiI (NsiI site is generated in the primer ΔE2A.DBP-start, underlined).

Left flanking sequences (corresponding Ad5 nucleotides 21557 to 22442):ΔE2A.DBP-stop: 5′-CCA ATG CAT ACG GCG CAG ACG G-3′ (SEQ ID NO:66)ΔE2A.BamHI: 5′-GAG GTG GAT CCC ATG GAC GAG-3′ (SEQ ID NO:67) Theamplified DNA fragment was digested with BamHI and NsiI (NsiI site isgenerated in the primer ΔE2A.DBP-stop, underlined).

Subsequently, the digested DNA fragments were ligated into SnaBI/BamHIdigested pBr/Ad.Sal-rITR. Sequencing confirmed the exact replacement ofthe DBP coding region with a unique NsiI site in pBr/Ad.Sal-rITRΔE2A.The unique NsiI site can be used to introduce an expression cassette fora gene to be transduced by the recombinant vector.

The deletion of the E2A coding sequences was performed such that thesplice acceptor sites of the 100K encoding L4-gene at position 24048 inthe top strand was left intact. In addition, the poly adenylationsignals of the original E2A-RNA and L3-RNAs at the lefthand site of theE2A coding sequences were left intact. This ensures proper expression ofthe L3-genes and the gene encoding the 100K L4-protein during theadenoviral life cycle.

Next, the plasmid pWE/Ad.AflII-rITRΔE2A was generated. The plasmidpBr/Ad.Sal-rITRΔE2A was digested with BamHI and SpeI. The 3.9 kbfragment in which the E2A coding region was replaced by the unique NsiIsite was isolated. The pWE/Ad.AflII-rITR was digested with BaniHI andSpeI. The 35 kb DNA fragment, from which the BamHI/SpeI fragmentcontaining the E2A coding sequence was removed, was isolated. Thefragments were ligated and packaged using λ phage-packaging extractsaccording to the manufacturer's protocol (Stratagene), yielding theplasmid pWE/Ad.AflII-rITRΔE2A.

This cosmid clone can be used to generate adenoviral vectors that aredeleted for E2A by cotransfection of PacI digested DNA along withdigested adapter plasmids onto packaging cells that express functionalE2A gene product. Examples of E2A complementing cell lines are describedbelow:

Generation of pBr/Ad.Bam-rITRsp and pWE/Ad.AflII-rITRsp

The 3′ ITR in the vector pWE/Ad.AflII-rITR does not include the terminalG-nucleotide. Furthermore, the PacI site is located almost 30 bp fromthe right ITR. Both these characteristics may decrease the efficiency ofvirus generation due to inefficient initiation of replication at the3′ITR. Note that during virus generation, the left ITR in the adapterplasmid is intact and enables replication of the viral DNA afterhomologous recombination.

To improve the efficiency of initiation of replication at the 3′ITR, thepWE/Ad.AflII-rITR was modified as follows: constructpBr/Ad.Bam-rITRpac#2 was first digested with PacI and then partiallydigested with AvrII. The 17.8 kb vector containing fragment was isolatedand dephophorylated using SAP enzyme (Boehringer Mannheim). Thisfragment lacks the adenoviral sequences from nucleotide 35464 to the 3′ITR. Using DNA from pWE/Ad.AflII-rITR as a template and the primersITR-EPH: 5′-CGG AAT TCT TAA TTA AGT TAA CAT CAT CAA TAA TAT ACC-3′ (SEQID NO:68) and Ad101: 5′-TGA TTC ACA TCG GTC AGT GC-3′ (SEQ ID NO:69), a630 bp PCR fragment was generated corresponding to the 3′ Ad5 sequences.This PCR fragment was subsequently cloned into the vector pCR2.1(Invitrogen) and clones containing the PCR fragment were isolated andsequenced to check correct amplification of the DNA. The PCR clone wasthen digested with PacI and AvrII. The resulting 0.5 kb adeno insert wasligated to the PacI/partial AvrII digested pBr/Ad.Bam-rITRpac#2fragment, generating pBr/Ad.Bam-rITRsp. Next, this construct was used togenerate a cosmid clone that has an insert corresponding to theadenosequences 3534 to 35938. This clone was named pWE/AflII-rITRsp.

Generation of pBr/Ad.Bam-rITRspΔXba and pWE/Ad.AflII-rITRspΔXba

Plasmid pBr/Ad.Bam-rITRsp was propagated in E. coli strain DM1 (dam⁻;dcm⁻) (Life Technologies). The plasmid was digested with XbaI, removingthe 1.88 kb XbaI-XbaI fragment, and religated. The resulting clone,pBr/Ad.Bam-rITRspΔXba, was used to construct helper cosmidpWE/Ad.AflII-rITRspΔXba as described above. The following fragments wereisolated by extraction from an agarose gel (QIAGEN): pWE.pac digestedwith PacI, pBr/AflII-Bam digested with PacI and BamHI, andpBr/Ad.Bam-rITRΔXba digested with BamHI and PacI. These fragments wereligated together and packaged using lambda phage packaging extractsaccording to the maufacturer's instructions (Stratagene). Afterinfection of host bacteria assembled phage, the resulting colonies wereanalyzed for the presence of the intact insert. pWE/Ad.AflII-rITRspΔXbacontains sequences identical to that of pWE/Ad.AflII-rITRsp, but withdeletion of the XbaI-XbaI fragment.

Generation of pBr/Ad.Bam-rITRspΔE2AΔXba and pWE/Ad.AflII-rITRspΔE2AΔXba

Plasmid pBr/Ad.Bam-rITRspΔE2AΔXba was constructed for the generation ofE1-deleted recombinant adenoviruses with dual deletion of E2A and E3. ASpeI-BamHI fragment containing E2A deletion was isolated from plasmidpBr/Ad.Sal-rITRΔE2A and inserted into SpeI-BamHI-digestedpBr/Ad.Bam-rITRspΔXba, yielding plasmid pBr/Ad.Bam-rITRspΔE2AΔXba. Thisplasmid was used to construct helper pWE/Ad.AflII-rITRspΔE2AΔXba, usingthree fragment ligation as described above. pWE/Ad.AflII-rITRspΔE2AΔXbacontains sequences identical to that of pWE/Ad.AflII-rITRsp, but withdual deletions of the E2A region and the XbaI-XbaI fragment.

Generation of pBr/Ad.Bam-rITRspΔE3, pWE/Ad.AflII-rITRspΔE3,pBr/Ad.Bam-rITRspΔE2AΔE3, and pWE/Ad.AflII-rITRspΔE2AΔE3

To allow insertion of larger DNA fragments, an expanded deletion of theE3 region was constructed in which the complete E3 coding region wasremoved. Primers 1 (5′-AAA CCG AAT TCT CTT GGA ACA GGC GGC-3′)(SEQ IDNO: 1) and 2 (5′-GCT CTA GAC TTA ACT ATC AGT CGT AGC CGT CCG CCG-3′)(SEQID NO:2) were used to amplify a sequence from pBr/Ad.Bam-rITRsp,corresponding to sequence 27326 to 27857 in wt Ad5 genome. Primers 3(5′-GCT CTA GAC CTC CTG TTC CTG TCC ATC CGC-3′) (SEQ ID NO:3) and 4(5′-GTA TGT TGT TCT GGA GCG GGA GGG TGC-3′) (SEQ ID NO:4) were used toamplify the sequence from the same DNA template, corresponding tosequences 30994 to 35502 in wt Ad5 genome. The amplification productswere digested with EcoRI/XbaI and XbaI/AvrII, respectively, and ligatedtogether. The resulting EcoRI-AvrII fragment was cloned into vectors ofpBr/Ad.Bam-rITRsp and pBr/Ad.Bam-rITRspΔE2A that had been digested withEcoRI and AvrII, yielding pBr/Ad.Bam-rITRspΔE3 and pBr/Ad.Bam-rITRspΔE2AΔE3, respectively. These two plasmids were used to construct cosmidhelper molecules as described above. pWE/Ad.AflII-rITRspΔE3 containssequences identical to sequences of pWE/Ad.AflII-rITRsp, but with adeletion of the E3 region corresponding to sequences 27857–30994 in wtAd5 genome. pWE/Ad.AflII-rITRspΔE2ΔAdE3 is identical topWE/Ad.AflII-rITRspΔE3, but with an additional deletion of the E2Aregion.

The above-described cosmids are particularly useful for the productionof adenoviral expression libraries, in particular, libraries carryingcollections of large inserts. See also Examples 19 and 20.

Example 19 Miniaturized, Multiwell Production of E1, E2A and E3 DeletedRecombinant Adenoviral Vectors Carrying Therapeutic and MarkerTransgenes

As mentioned in Example 10, a combined deletion of E1, E2A, and E3 willallow cloning of foreign DNA sequences up to approximate 10.5 kb insize. Here, the production of E1, E2A, and E3 deleted vectors carryinghuman cDNAs is shown, as well as marker genes in PER.C6/E2A cells.

Cell culture conditions were described in Example 10. For DNAtransfection, adapter and helper molecules were prepared according toExample 14. Linearized adapter plasmids pAD/CLIP-ceNOS and pAD/CLIP-lacZwere used for transfection in combination with four differentPacI-linearized helper cosmids, namely pWE/Ad.AflII-rITRsp,pWE/Ad.AflII-rITRsp.dE2A, pWE/Ad.AflII-rITRsp.dXba, andpWE/Ad.AflII-rITR. The DNA transfection procedure was identical to thatdescribed in Example 10. An aliquot of 100 μl of the freeze/thawedlysates was used to infect a second 96-well plate with PER.C6/E2A cellsand CPE formation was monitored. FIG. 35 shows the percentage of virusproducing wells (CPE positive) in a 96-well plate of PER.C6/E2A cellafter propagation of the freeze/thawed transfected cells. Clearly, it ispossible to produce E1 and E3 deleted recombinant adenoviral vectorscarrying therapeutic and marker transgenes in PER.C6/E2A cells.

Example 20 Construction of a Sense or Antisense, Arrayed AdenoviralExpression Library for Selecting Phenotypes

The miniaturization of adenoviral vector production allows thelarge-scale, high throughput construction, screening of cloned or pooledgene expression libraries.

To construct a cloned and arrayed cDNA expression library in anadenoviral vector format based on the PER.C6 (and derivatives)production system, poly(A+) mRNA of human placenta is isolated usingoligo(dT) cellulose and converted into cDNA using materials and reagentssupplied by vendors such as Life Technologies Inc. (LTI; Breda, NL). Theresulting double stranded cDNA molecules contain a SalI-compatibleoverhang at the 5′ end and a NotI-compatible overhang at the 3′ end. Thetotal cDNA was ligated into pIPspAdapt6 (for sense orientation of cDNAinserts ) or pIPspAdapt7 (for antisense orientation of cDNA inserts)(see example 17 for adapter configurations). Then, pIPspAdapt6 andpIPspAdapt7 were digested with the restriction endonuclease MluI,followed by dephosphorylation of the 5′ overhangs using thermosensitivealkaline phosphatase (LTI). After digestion with SalI and NotI, thelinearized plasmid was isolated on a 0.8% agarose gel and purified byanion exchange chromatography. Following ligation of the cDNA moleculesinto the plasmids, the resulting library was introduced into E. coliDH5a electrocompetent bacteria by electroporation on a BTX 600electrocell manipulator or equivalent. The unamplified library wasaliquoted and frozen as a glycerol stock.

On the day of plating, vials were thawed and plated on large petridishes containing LB media with 1.5% agar and ampicillin at 50micrograms per ml. To obtain even distribution of the plated colonies,glass beads were used while plating. After overnight growth at 37° C.,the agar-plates were transferred to an automated colony-picking robot(Flexys; Genome solutions). Individual colonies were picked andtransferred by the robot to microtiter plates with 300 μl of TerrificBroth media and ampicillin at 50 micrograms per ml. Plates inoculated inthis way are then transferred to HiGro incubators (Genemachines) aeratedwith oxygen and grown according to the manufacturer's manual for 12–16hours. Thereafter, the individual plasmids were isolated by theconventional alkaline lysis plasmid DNA isolation method as described inSambrook et al. (Sambrook, J. et al, eds. (1989) Molecular cloning: alaboratory manual, 2^(nd) edition, Cold Spring Harbor Laboratory Press).For this, the plates are first transferred to centrifuges (e.g.,Eppendorf 5810R or Heraeus Megafuge 2.0) and bacteria are pelleted for20 minutes at 1500×g. Using robotic liquid handlers, the supernatant inthe individual wells of the individual plates is removed and discarded.Bacterial pellets are resuspended in 100 μl 25 mM Tris, pH 8.0containing 50 mM glucose and 10 mM EDTA, and bacteria are lysed byadding 100 μl of 0.2N NaOH/1% SDS. Following neutralization by adding100 μl of 5M potassium acetate, a cleared lysate is obtained byfiltration over a MultiScreen-NA lysate clearing plate (Millipore B.V.,Etten-Leur) or equivalent thereof, using a vacuum manifold. The plasmidDNA in the cleared lysate is subsequently precipitated by adding 200 μlof 2-isopropanol and centrifugation (1500×g, 30 minutes, 4° C.). Theprecipitate is washed once with 70% ethanol, air dried, and resuspendedin 20 μl of TE.

The isolated plasmid DNA in each individual well is quantified using thePicogreen DNA quantification kit (Molecular Probes, Eugene, Oreg. USA)by transferring an aliquot of the plasmid DNA from each well to freshplates with the appropriate dilution. PER.C6 cells, or derivatives suchas PER.C6/E2A, are seeded as described under the other examples forminiaturized adenovirus generation. For each well, 55 nanogram ofpurified plasmid DNA was transferred into a new plate and linearizedwith PiPspI for 60 minutes at 65° C. This plasmid is then cotransfectedwith an appropriate helper DNA molecule (e.g., E2A deleted (such aspWE/Ad.AflII-rITR.deltaE2A), E2A/E3 deleted, or E2A/E3/E4 deleted, seeexample 18) into PER.C6 or PER.C6/E2A packaging cells. Transfection issimilar to the experiments and methods described in examples 9, 10, or25 for adenovirus generation in microtiter plates. Viral formation inindividual wells is quantified using CPE formation, blot based virusassays, or reporter systems. The arrayed adenoviral library is thenready to be used in cell based screens where one can select for aparticular phenotype.

In FIG. 36A and FIG. 36B, an overview is given of the scheme of anadenoviral cDNA expression library constructed and arrayed as describedabove. This scheme describes the construction of libraries ofindividually cloned adenoviral vector libraries in a high throughputfashion. The improvement of this strategy over pooled libraries is thatno bias for viruses with a growth advantage can occur. This is becauseindividual members of the library are in the format of individualcolonies straight after the plating of the library, and are keptindividually during all further procedures.

The adenoviral expression library can be used for infection of differentcells appropriate for selection of a particular phenotype such ascapillary formation, cell proliferation, cell migration, or marker geneexpression either in an appropriate unmodified cell type or a reportercell line designed for this purpose. Detection of these phenotypes canbe done, for example, using automated image analysis of morphologychanges or changes in intracellular localization of a reporter protein.Once hits have been selected, the cloned bacterial DNA version isavailable immediately for sequence analysis in the form of pIPspAdapt6or pIPspAdapt7 adapter plasmid as produced in E. coli. This means thatno rescue is necessary, as is the case with pooled retroviral orplasmid-based expression libraries.

If desired (for example, using robotic liquid handlers or manually),individual wells containing individual adenoviral vectors in one row,one column, one plate, or multiple plates can be pooled before doingassays. This may be advantageous if a desired assay is not amenable tohigh throughput analyses and the total number of wells needs to bedecreased for a primary screen. An additional improvement or advantageof pooled but originally cloned adenoviral vectors is that multi-genedependant phenotypes are selectable.

Example 21 Viral Production in Wells of a 384-well Tissue Culture Plate

Essentially, this experiment was performed as described in Example 10,except for the following minor changes. The day before transfection,PER.C6/E2A cells were diluted with culture medium (DMEM with 10% fetalbovine serum and 10 mM MgCl₂) to a suspension of 11,250 cells per 25 μl,followed by seeding 25 μl per well of a 384-well tissue culture plateusing a 16 channel multichannel pipette (Finn). After adding 1.3 mlserum free DMEM to the DNA/lipofectamine mixture, 15 μl of this mixturewas then added to each PER.C6/E2A seeded well that had been washed with25 μl DMEM prior to transfection. After 3 hours in a humidified CO₂incubator (39° C., 10% CO₂), 50 μl culture medium was added to each welland the plates were returned to the humidified CO₂ incubator (39° C.,10% CO₂). The next day, the medium of each well was replaced with 50 μlculture medium. The plates were then returned to a humidified CO₂incubator (32° C., 10% CO₂) for an additional 4 days, after which thewells were subjected to freezing at −20° C. overnight followed bythawing and resuspension by repeated pipetting. An aliquot of 25 μl ofthe freeze/thawed transfected cells was transferred to each well of aplate with fresh PER.C6/E2A cells seeded as described above on 384-welltissue culture plates (plate 2). The second 384-well plate, withPER.C6/E2A cells incubated and thus infected with freeze/thawed celllysate of the first transfected plate, was checked for CPE formation andstored at −20° C. The experiment mentioned above was performed twice. InFIG. 37A, FIG. 37B, FIG. 37C, and FIG. 37D, the percentage of CPEpositive wells scored after propagation of the freeze/thawed transfectedcells to new PER.C6/E2A cells is depicted.

Example 22 The Effect of Omitting Propagation, or Refreshment of CultureMedium Instead of Propagation, on the Speed and Production Efficiency ofVirus Formation

Making the process of miniaturized adenoviral vector production moreamenable to automation calls for a simplification of the wholeprocedure. One laborious and time consuming step is the propagation ofcell lysates from transfected PER.C6/E2A cells on fresh cells, thereforeomitting this step is desirable. In order to determine the effect ofchanging the medium of transfected cells instead of using thefreeze/thawed, transfected PER.C6/E2A cells (see example 10 and others)to infect new PER.C6/E2A cells (propagation), or to omit propagation alltogether, the following experiment was performed. The day beforetransfection, PER.C6/E2A cells were harvested using trypsin-EDTA andthen counted. The cells were then diluted with culture medium (DMEM with10% fetal bovine serum and 10 mM MgCl₂) to a suspension of 22,500 cellsper 100 μl, followed by seeding 100 μl per well in the 96-well tissueculture plates. The next day, 2.6 μg of the linearized adapter moleculesand 2.6 μg of the PacI linearized pWE-Ad.AflII-rITRdE2A plasmid DNA, ina volume of 100 μl serum free DMEM, were mixed with 26.5 μllipofectamine diluted in 74.4 μl serum free DMEM by adding thelipofectamine mix to the DNA mix. The DNA/lipofectamine mixture was leftat room temperature for 30 minutes, after which 1.3 ml serum free DMEMwas added. The mixture was then added (30 μl per well) to PER.C6/E2Aseeded wells that had been washed with 200 μl DMEM prior totransfection. All of the transfections were performed in duplicate.After three hours in a humidified CO₂ incubator (39° C., 10% CO₂), 200μl culture medium was added to each well and the plates were returned tothe humidified CO₂ incubator (39° C., 10% CO₂). The next day, the mediumof each well was replaced with 200 μl culture medium. The plates werethen returned to the humidified CO₂ incubator (32° C., 10% CO₂). Afterseven days, the medium of one of the two transfected plates was replacedwith 200 μl culture medium, returned to the humidified CO₂ incubator(32° C., 10% CO₂), and the formation of CPE was followed. In FIG. 38A,the percentage of virus producing cells (CPE positive wells) scoredafter changing the medium of the transfected cells, instead ofpropagation and amplification fresh PER.C6/E2A cells, is depicted.

The wells of the second plate were subjected to freezing at −20° C.overnight, followed by thawing and resuspension by repeated pipetting. A100 μl aliquot of the freeze/thawed transfected cells was transferred toeach well of a plate with new PER.C6/E2A cells (2.25×10⁴ cells per wellin 100 μl) that had been seeded in 96-well tissue culture plates one dayprior to infections. The plate was incubated in the humidified CO₂incubator (32° C., 10% CO₂) until the presence of full CPE was observed.In FIG. 38B, the percentage of virus producing cells (CPE positivewells) scored after propagation on freshPER.C6/E2A cells is depicted. Inall experiments, untransfected wells were included for control of crosscontamination. All the control wells remained negative for CPEformation. In FIG. 38C, the results of a parallel normal procedure asdescribed under example 10 are shown.

These results show that replacement of medium or completely omitting anyhandling after transfection can replace reinfection of fresh PER.C6/E2Acells with lysate from the primary transfectant plates.

Example 23 Determination of the Influence of the Cell Growth ofPER.C6/E2A Cells on the Speed and Production Efficiency of VirusFormation

For construction of adenoviral gene expression libraries, the conditionsfor miniaturized production of adenoviral vector need to be optimal.Therefore, a number of parameters that may influence virus generationwere varied and their effects in adenoviral vector production weremeasured.

In order to determine whether the cell confluency of the complementingcell line PER.C6/E2A, prior to seeding in microtiter plates, influencesthe speed and efficiency of virus production, the following experimentwas performed. On day one, PER.C6/E2A cells were harvested usingtrypsin-EDTA and counted. The cells were seeded at 1/10, 1/5, and 1/2.5of the harvested cells in three different 175 cm² tissue culture flasks.In table 9, the number of cells that were seeded in each 175 cm² tissueculture flask in three different experiments are shown. Four days later,the PER.C6/E2A cells from each flask were harvested, counted, and thendiluted with culture medium (DMEM with 10% fetal bovine serum and 10 mMMgCl₂) to a suspension of 22,500 cells per 100 μl. From each cellsuspension, two 96-well tissue culture plates were seeded with 100 μl ofcell suspension per well. The next day, 10.6 μg of SalI linearizedpAd/Clip-lacZ and 10.6 μg of the PacI linearized pWE-Ad.AflII-rITRdE2Aplasmid DNA, in a volume of 600 μl serum free DMEM, were mixed with153.6 μl lipofectamine diluted in 446.4 μl serum free DMEM by adding thelipofectamine mix to the DNA mix. The DNA/lipofectamine mixture was leftat room temperature for 30 minutes, after which 7.8 ml serum free DMEMwas added. The latter mixture was then added (30 μl per well) toPER.C6/E2A seeded wells that had been washed with 200 μl DMEM prior totransfection. After three hours in a humidified CO₂ incubator (39° C.,10% CO₂), 200 μl DMEM with 10% fetal bovine serum and 10 mM MgCl₂ wasadded to each well and the plates were returned to the humidified CO₂incubator (39° C., 10% CO₂). The next day, the medium of each well wasreplaced with 200 μl DMEM with 10% fetal bovine serum and 10 mM MgCl₂.The plates were then returned to a humidified CO₂ incubator (32° C., 10%CO₂). After two days, one of the two transfected plates was used todetermine the transfection efficiency using lacZ staining. Thetransfection efficiency of each 96-well tissue culture plate, scoredafter lacZ staining in three different experiments, is shown in Table 9.

The second plate of the two transfected plates was used for virusproduction. Seven days after transfection, the wells of the second platewere subjected to freezing at −20° C. overnight, followed by thawing andresuspension by repeated pipetting. A 100 μl aliquot of thefreeze/thawed transfected cells was transferred to each well of a platewith new PER.C6/E2A cells (2.25×10⁴ cells per well in 100 μl) that wereseeded in 96-well tissue culture plates one day prior to infections. Theplate was incubated in the humidified CO₂ incubator (32°, 10% CO₂) untilthe presence of full CPE was observed. In FIG. 39A, FIG. 39B, and FIG.39C, the percentage of virus producing cells (CPE positive wells),scored after propagation of the freeze/thawed transfected cells to newPER.C6/E2A cells, is depicted. The data indicate that the level ofconfluency of the PER.C6/E2A cells, prior to transfection with theadenoviral adapter and helper DNA molecules, influences the finalpercentage of virus producing wells. The higher confluency was the mostoptimal for absolute fmal number of wells producing virus and the speedat which the virus generation occurs.

Example 24 Long-term Incubation with Adenoviral Supernatant AllowsDetection of Slow Phenotypes

The use of adenoviral vector libraries in functional genomics calls forthe use of appropriate cell based assays which are amenable to highthroughput screening and miniaturization, in addition to a phenotypethat is detectable and relevant for the genes one is looking for, suchas the ones used in example 12. The time of assaying after infectionwith an adenoviral expression library, for example as described inExample 20, is variable and dependent on the parameters determined bythe phenotype being assayed for. For example, using automated imageanalysis, the formation of blood capillaries in each well can be assayedsimply by detecting the formation of capillaries. Formation of thesestructures, which are indicative of angiogenesis or blood vesselformation, can be induced by infection of relevant precursor cells. Suchcells can be endothelial cells from heart or tumor origin with anadenoviral vector carrying a relevant transgene, for example a vascularendothelial like growth vector (VEGF). However, a complex phenotype suchas capillary formation only appears after several days to weeks.Therefore, expression of the library of genes as mediated by theadenoviral expression cassette in some cases needs to be long enough toallow the phenotype to develop. In FIG. 40, the results of an experimentwith an EGFP-adenoviral vector that was used to infect A549 cells in96-well plates are shown. Based on the stability features described inexample 15, the adenoviral dilution (in DMEM) was not removed but leftfor up to 2 weeks and EGFP expression measured at regular intervals.Clearly, these experiments show that adenoviral transduction can beregarded as semi-stable and even increase over time, suggesting thatreinfection occurs and/or that newly divided cells are infected. Thisimplies that the transient adenoviral vector system can be used toscreen for phenotypes that take 2 weeks or more to develop by leavingthe adenoviral supernatant on the cells in the multiwell plates(384-well or smaller).

Example 25 Miniaturized, Multiwell Production of Recombinant AdenoviralVectors Using Cost Effective Polyethylenimine (PEI) as DNA TransfectionAgent

For the purpose of cost reduction and variable toxicity reduction, it isdesirable to replace the liposomal transfection reagent lipofectamine.The cationic polymer polyethylenimine (PEI) has been tested in theminiaturized, multiwell (96-well) adenoviral vector production systemfor this purpose. See also Examples 9 and 10. PEI has been tested fortransfection of PER.C6 and PER.C6/E2A, with different transgene insertsin the adenoviral helper plasmid: LacZ and EGFP. Different parameterswere tested including PEI/DNA ratios, incubation times, and amounts ofPEI/DNA complex per single well.

Testing of PEI with Different PEI/DNA Ratios

The 96-well microtiter plates were seeded the day before transfectionwith PER.C6 or PER.C6/E2A cells as described in example 10. Then, 3micrograms of SalI linearized pCLIP-LacZ and 3 micrograms PacIlinearized pWEAflIIrITR for PER.C6 or pWEAflIIrITRdE2A for PER.C6/E2Awere diluted in 150 μl 150 mM NaCl and incubated at room temperature for10 minutes. Also, a 20 mM 25 kDa PEI solution was diluted in 150 μl of150 mM NaCl at different amounts to obtain different PEI/DNA ratio's andincubated at room temperature for 10 minutes. See table 4. The assayswere performed on sixteen wells (8 wells in duplicate on differentplates).

The DNA and PEI solutions were mixed by adding PEI dropwise to DNA andthen incubated for 10 minutes at room temperature. Cells were washedwith 100 μl of serum free DMEM per well. Then, 1.3 ml of DMEM was addedto the mixture and 80 μl of the solution was applied to the cells ineach well. As a positive control, DNA/lipofectamine complexes weretransfected (prepared according to Example 9). Additional controlincubations of DMEM, PEI without DNA (ratio 13), and two times theamount of PEI/DNA ratio 13 were included. The transfections were donefour hours later. For the PEI transfections, 80 μl of PEIPER.C6 medium(DMEM with 20% v/v fetal calf serum (FCS) 10 mM MgCl₂)/well was added tothe cells. For the lipofectamine transfections, 180 μl of DMEM, 10% v/vFCS, 10 mM MgCl2 per well was added to the cells. The plates werereturned to a humidified CO₂ incubator and incubated at 37° C. forPER.C6 and at 39° C. for PER.C6/E2A. The next day, the medium of eachwell was replaced with 200 μl DMEM, 10% v/v FCS, 10 mM MgCl2. The plateswere then left at 37° C. for PER.C6 plates and at 32° C. for PER.C6/E2Aplates in a humidified CO₂ incubator. After 3 days, one of the duplicateplates was stained with X-gal to determine the transfection efficiency,the results of which are depicted in table 5.

After four days post-transfection, the plates were subjected to freezingat −20° C. for 4 hrs, followed by thawing and resuspension by repeatedpipetting. A 100 μl aliquot was transferred to a new plate of PER.C6 orPER.C6/E2A cells seeded a day before as described above. The plates werethen placed back into the CO₂ incubators. After 14 dayspost-propagation, virus formation was scored by CPE as an indicator andthe plates were subjected to freezing at −20° C. for 4 hrs, followed bythawing and resuspension by repeated pipetting. A 20 μl aliquot wastransferred to wells of plates seeded with 1×10⁴ A549 cells per well ofa 96-well plate in a volume of 100 μl. Two days after infection, thewells were stained with X-Gal for LacZ activity as described underexample 9. The results are summarized in table 6.

Testing of PEI with Different PEI/DNA Ratios in Combination withDifferent Amounts of Complex Per Well

In order to test the optimal amount of PEI/DNA complex that can beapplied to the cells without being toxic, the PEI/DNA ratios 5 and 11.7were tested on PER.C6/E2A. The standard concentration (1X) is theconcentration as described in the previous transfection experiment (seeTable 4).

To make PEI solutions with amounts of PEI between 0.9 and 42 μl, variousamounts of a 150 mM NaCl solution were added to the 20 mM 25 kDa PEI(Fluka cat.nr.03880) solution to a final volume of 300 ul. From thissolution, 150 μl was added to the DNA mix (see table 7). DNA (50%pCLIP-LacZ and 50% pWEAflIIrITRdE2A) to 150 microliters with 150 mMNaCl.

Transfections were performed as described above. Lipofectamine was usedas a positive control, as well as DNA, PEI, or DMEM without anyadditives. After three days, a duplicate plate was stained for lacZexpression. These results are given in table 8. First, the cells werechecked for toxicity. At ratio 11.7, the double concentration (2X) ismore toxic but the transfection efficiency at X is higher. Atconcentration 0.1X for both ratios, no blue cells were seen afterstaining, indicating that the cells were not transfected. Processing,CPE monitoring, A549 transduction, and lacZ staining was done asdescribed.

To quantitatively test toxicity, the latter transfections were repeated.Two days after medium replacement, a cell proliferation assay (Promega)was used to determine the number of living cells or toxicity of PEI/DNAcomplexes. All actions were according to the manufacturer's protocol.After 4 hrs of incubation at 37° C., the plates were read using themicroplate manager (Bio-Rad). The results of this experiment for PEIratio 5 and 11.7 are summarized in FIG. 41. Clearly, toxicity is lowestand virus generation optimal at ratio 5 (1.5 times the standard amountof complex) and at ratio 11.7 (at the standard conditions between 0.5and 1.5 times the standard amounts).

Testing PEI as a DNA Carrier with Different PEI/DNA Ratios, with aDifferent Gene, and at Different Temperatures

In order to determine if the temperature of PEI influences the complexformation, the above-described protocol was tested with PEI at 4° C. andat room temperature. The best concentrations of the two ratios were usedin this transfection experiment (450 ng DNA/well PEI/DNA ratio 5 and 300ng DNA/well PEI/DNA ratio 11.7). In addition, another transgene insert,EGFP, was tested. Processing, CPE monitoring, A549 transduction, andlacZ staining were performed as described. As can be seen in FIG. 42,there is no significant difference between warm and cold PEI. Virusformation with EGFP and PEI worked very well (PEI ratio 5 warm and thepositive control lipofectamine both 100% CPE).

Testing of PEI as DNA Carrier with Different PEI/DNA Complex Volumes PerWell

In order to test if the volume of DNA/PEI complex influenced theefficiency of virus generation in the above-described protocol, 30, 80,and 120 μl per well of the PEI/DNA complex (ratio 5 450 ng DNA per well,PEI 20 mM 25 kDa, Fluka) were added to the cells. Processing, CPEmonitoring, A549 transduction, and lacZ staining were done as describedabove. As is shown in FIG. 43, there is a significant difference intransfection efficiency between applying 30 μl, 80 μl, and 120 μl. Only1% of the cells within a well were stained blue with 30 μl, whereas 60%of the cells stained blue with 80 μl. The 120 μl sample showed the sameresults as applying 80 μl. For virus formation, the same trend wasobserved: no CPE was found for 30 μl, whereas 80 μl and 120 μl gavesimilar percentages (not shown). In conclusion, PEI can be used toproduce adenoviral vectors in a miniaturized setup.

Example 26 Miniaturized, Multiwell Production of Recombinant AdenoviralVectors Without a Cell Washing Step Prior to Transfection

In order to reduce steps in automation of the miniaturized, multiwellproduction of recombinant adenoviral vectors and cost reduction, theserum free medium (SFM) washing step of the PER.C6 or PER.C6/E2A cellsor derivatives prior to transfection was removed from the standardprotocol. Transfections were performed as described in example 10. Thetransfections were performed using the human ceNOS as the transgeneinsert. Removal of the cell washing step was tested and compared to thestandard procedure. Processing and CPE monitoring were done asdescribed. As seen in FIG. 44, there is no significant reduction invirus production when the cells were not washed prior to transfection.

In conclusion, removal of the cell washing step, which removes the bulkof serum proteins as part of the standard transfection protocol, ispossible without effecting the CPE efficiency. Removal of this step isvery useful when reducing the complexity of the whole process isdesirable for automating the miniaturized, multiwell production ofrecombinant adenoviral vectors.

Example 27 The Use of Adenoviral Constructs to Modulate Gene Expressionin Zebrafish

Modulation of gene expression by adenoviral constructs in whole animalscan give important information about the function of genes. Forinstance, adenoviral constructs that express a sense cDNA constructencoding a full length protein can be used for overexpression of thatprotein in animal model systems, while adenoviral constructs thatexpress the antisense cDNA can be used to reduce the expression levelsof the endogenous protein. In addition, overexpression of anadenoviral-encoded protein might rescue a mutant phenotype.Adenoviral-mediated modulation of gene expression in animal models cangive important information about the function of a gene. In thisexample, zebrafish, Danio rerio, will be discussed as an animal modelsystem to show the feasibility of the approach. Zebrafish cDNA librarieswill be screened with cDNAs that are identified and isolated by methodsdescribed in this application. The obtained homologous zebrafish cDNAs,encoding full length proteins, will be isolated and cloned in bothorientations, sense and antisense, in adapter plasmids of the pIPSPAdaptseries (see example 17). These will subsequently be used to generaterecombinant adenovirus, which will be used to infect either wildtype ormutant zebrafish embryos (see for instance Development 1996 Volume 123,December). Methods for breeding zebrafish are well known to thoseskilled in the art.

The effect of up- or down-modulation of gene expression can be studiedin wildtype or mutant embryos or adult fish. Embryos will be collectedas follows: zebrafish are photo-periodic in their breeding and produceembryos every morning shortly after sunrise. For continuous productionof a relatively small number of embryos (30–50 per tank per day), anequal number of males and females are used. The day-night cycle iscontrolled with an automatic timer (14 hr light/10 hr dark). The bottomof the tank is covered with a single layer of marbles to keep the fishfrom eating the newly spawned eggs. Freshly produced embryos arecollected each morning by siphoning the bottom of the tank. The embryoscan then be infected with recombinant adenovirus in several ways, asdescribed below. The method of choice depends on the expression patternof the gene.

Recombinant adenovirus can be injected directly into the chorion fluid,after which the embryos are washed and cultivated further in systemwater. Similarly, recombinant adenovirus can be deposited at specificsites in embryos or adult fish, for instance, by injection into theblood stream or by oral or rectal administration. Injection can beperformed by holding the embryos in wedged-shaped troughs made with aplastic mold in 1.5% agarose, in which case there is no need to removetheir chorions. Each trough can hold approximately 35 embryos (withchorions). Embryos can be aligned by gently tapping them down withforceps. Agarose is useful because pipette tips generally will not breakif they accidentally touch the surface. As the pipette penetrates thechorion, the embryo is forced against the rear vertical wall of thetrough. The exact positioning of the pipette tip within the embryo isachieved by slight movement of the pipette with a micro-manipulator orby movement of the stage. Alternatively, embryos can be dechorionated(see below) and incubated in medium containing recombinant adenovirus.

After injection, 25–30 eggs are deposited into 250 ml beakers. Afterhatching, larvae are transferred into a new beaker and completelyseparated from their chorions. Larvae are raised under standardconditions well known to those skilled in the art.

Monitoring changes after adenoviral infection of zebrafish can be doneas early as the embryonic stage. Some observations of zebrafishdevelopment can be made directly through the chorion. However, for mostprocedures it is better to remove the chorion, which is easily done withsharp forceps. When raised at 28.5° C., zebrafish develop normallyoutside their chorions. Embryos removed from their chorions can betransferred from one container to another by gently pipetting them upwith a fire-polished Pasteur pipette or by gentle pouring. Small petridishes (35 mm diameter) are adequate for holding up to 25 embryos duringthe first few days of development. The embryos can be brought to thecenter of the dish for viewing by gently swirling the medium in acircular motion. Larvae and adult fish can be monitored without furthertreatment.

More elaborate analysis methods include the staining of sections byclassical histological methods or specific methods such as anti-sensehybridization or incubation with antibodies to look at differences atthe molecular level.

The phenotypic changes observed after infection of zebrafish withrecombinant adenovirus can give important information about the functionof the encoded genes in vivo. The described method can also be appliedto other animal models.

TABLE 4 Ratio # 20 mM PEI (μl) # 150 mM NaCl (μl) 8.3 7.5 142.5 10 8 14211.7 10.5 139.5 13 12 138 15 13.5 136.5

TABLE 5 Transfection efficiency control. X-gal staining. Ratio % bluecells PER.C6 % blue cells PER.C6E2A   8.3 45 60 10 45 60   11.7 55 65 1355 65 15 50 40 2*13 10 10 Only PEI 13 0 0 Only DMEM 0 0 LIPO 65 80

TABLE 6 # blue % blue wells after wells after PER.C6 infection infectionRatio # CPE % CPE A549 A549   8.3 6/8 75 7/8 87.5 10 3/8 37.5 5/8 62.5  11.7 4/7 57 4/7 57 13 4/8 50 5/8 62.5 15 3/7 37.5 3/7 43 2*13 0/8 00/8 0 Only PEI 13 0/8 0 0/8 0 Only DMEM 0/8 0 0/8 0 Lipofectamine  1/166 1/8 12.5 # blue PER.C6/E2A wells A549 % blue Ratio # CPE % CPE cellsA549 cells   8.3 0/8 0 0/8 0 10 1/8 12.5 3/8 37.5   11.7 3/8 37.5 6/8 7513 1/8 12.5 2/8 25 15 1/8 12.5 2/8 25 2*13 0/8 0 0/8 0 Only PEI 13 0/8 00/8 0 Only DMEM 0/8 0 0/8 0 Lipofectamine 11/16 69 13/16 81

TABLE 7 Amount of Concentration of DNA/well PEI ratio PEI ratio PEI/DNAcomplex (ng/μl) DNA (μg) 11.7 (μl) 5 (μl) 2 X 600 12 42 18 1.5 X 450 931.5 13.5 Standard 1 X 300 6 21 9 0.5 X 150 3 10.5 4.5 0.1 X 30 0.6 2.10.9

TABLE 8 Amount of DNA % blue cells PEI % blue cells Complex (ng) perwell ratio 5 PEI ratio 11.7 PEI 2 X 600 30 45 PEI 1.5 X 450 40 55 PEIstandard X 300 25 Not determined PEI 0.5 X 150 5 40 PEI 0.1 X 30 0 0Lipofectamine 100 65 65 −/− 0 0 0

TABLE 9 T₁₇₅ flask Cell # 1^(st) exp. Cell # 2^(nd) exp. Cell # 3^(rd)exp. Confluencies of cells harvested for transfection 1/10 2.3 × 10⁶ 1.3× 10⁶  3.3 × 10⁶ 1/5 4.7 × 10⁶ 2.6 × 10⁶  6.7 × 10⁶ 1/2.5 — 5.2 × 10⁶13.3 × 10⁶ Efficiency 2^(nd) Efficiency 3^(rd) 96-well-plate Efficiency1^(st) exp. exp. exp. Transfection efficiencies 1/10 30–40% 50–60%50–60% 1/5 70–80% 50–60% 50–60% 1/2.5 — 50–60% 50–60%

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now having been fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

1. A method for determining the function of a unique nucleic acid present in a library, said method comprising: (a) providing a library of a multitude of unique expressible nucleic acids, said library including a multiplicity of compartments, each of said compartments consisting essentially of one or more adenoviral vector comprising at least one unique nucleic acid of said library in an aqueous medium, wherein said adenoviral vector is capable of introducing said nucleic acid into a host cell, is capable of expressing the product of said nucleic acid in said host cell, and is deleted in a portion of the adenoviral genome necessary for replication thereof in said host cell; (b) transducing a multiplicity of host cells with at least one adenoviral vector comprising at least one unique nucleic acid from said library; (c) incubating said host cells to allow expression of the product of said nucleic acid; and (d) determining the function of said nucleic acid.
 2. A method for determining at least one function of at least one nucleic acid present in a library of claim 1, said method comprising: transducing a multiplicity of cells with at least one vehicle comprising said at least one nucleic acid from said library, and culturing said multiplicity of cells while allowing for expression of said at least one nucleic acid and determining the expressed function thereof.
 3. The method of claim 1 wherein step (d) comprises observing said host cell to identify any changes in said host cell relative to a host cell that has not been transduced with an adenoviral vector comprising said nucleic acid.
 4. The method of claim 1, wherein the function of the expression product of all of said unique expressible nucleic acids in said library is unknown at the time said library is first made.
 5. The method of claim 1, wherein none of said compartments contain any adenoviral vector capable of replication except in a packaging cell containing said deleted portion of adenoviral genome.
 6. The method of claim 1, wherein said host cell is a eucaryotic cell.
 7. The method of claim 1, wherein at least one compartment comprises at least two adenoviral vectors.
 8. The method of claim 1, wherein each of said compartments consists essentially of one said adenoviral vector.
 9. The method of claim 5, wherein each of said compartments contains from about 0.01×10¹⁰ to about 10×10¹⁰ pfu of said adenoviral vector per ml of aqueous medium.
 10. The method of claim 9, wherein each of said compartments further contains the cellular debris from packaging cell lysate.
 11. The method of claim 5, wherein said adenoviral vector is a minimal vector.
 12. The method of claim 11, wherein said minimal vector comprises an adenovirus encapsidation signal or a functional part, derivative and/or analogue thereof, and at least one copy of at least a functional part or a derivative of an adenoviral ITR.
 13. The method of claim 5, wherein said adenoviral vector comprises adenoviral genomic sequence deleted for sequence encoding the E1-region proteins.
 14. The method of claim 12 wherein said minimal vector further comprises an adeno-associated virus terminal repeat or a functional part, derivative and/or analogue thereof.
 15. The method of claim 13, wherein said adenoviral vector is further deleted for sequence encoding the E2A-region proteins, or the E2B region proteins or the complete E2 region proteins.
 16. The method of claim 1, wherein said adenoviral vector further comprises adenovirus genomic sequence encoding adenoviral fiber proteins from at least two serotypes of adenovirus.
 17. The method according to claim 1, wherein said multiplicity of cells is divided over a multiplicity of compartments, each said compartment comprising at least one vector.
 18. The method according to claim 1, further comprising selecting at least one vector having a desired function.
 19. The method according to claim 1, wherein at least one of said performed steps is automated.
 20. A method for obtaining an expressible nucleic acid having a desired function when expressed in a cell, said method comprising: (a) performing the method of claim 1; (b) determining which compartment in said library contains an adenoviral vector comprising a unique nucleic acid having said desired function; and (c) obtaining said vector from said compartment.
 21. The method according to claim 1, wherein said multiplicity of compartments comprises a multiwell format of at least 6 wells.
 22. The method according to claim 21, wherein substantially each said well consists essentially of one or more said adenoviral vector comprising said unique nucleic acid that encodes a product of unknown function.
 23. The method according to claim 21, wherein said library is configured to be made and used in a substantially automated process.
 24. The method according to claim 21, wherein said multiplicity of compartments comprises a multiwell format of at least 96 wells.
 25. The method according to claim 24, wherein each well contains cellular debris from eucaryotic packaging cell lysate.
 26. The method of claim 24, wherein none of said wells contains adenoviral vector capable of replication except in a packaging cell containing said deleted portion of adenoviral genome.
 27. The method of claim 26, wherein each of said wells contains from about 0.01×10¹⁰ to about 10×10¹⁰ pfu of said adenoviral vector per ml of aqueous medium.
 28. The method of claim 1, wherein each of said unique nucleic acids is derived from a member of a population of nucleic acids, said population selected from the group consisting of naturally occurring populations of messenger RNA, DNAs, cDNAs, genes, ESTs and genetic suppressor elements; synthetic oligonucleotides; and antisense nucleic acids.
 29. The method of claim 10, wherein the contents of each said compartment is capable of transfecting said host cell and expressing the product of each said unique nucleic acid in said host cell.
 30. The method of claim 29, wherein each said compartment is capable of providing from about 400 to about 4000 aliquots of said adenoviral vector.
 31. The method of claim 27, wherein the contents of each said well is capable of transfecting said host cell and expressing the product of each said unique nucleic acid in said host cell.
 32. The method of claim 31, wherein each said well is capable of providing from about 400 to about 4000 aliquots of said adenoviral vector.
 33. The method of claim 12, wherein said minimal vector comprises an regulatable promoter operably linked to said unique nucleic acid.
 34. The method of claim 13, wherein said adenoviral vector comprises an regulatable promoter operably linked to said unique nucleic acid.
 35. The method of claim 13, wherein said adenoviral vector is further deleted for the adenoviral E3-region or a functional part thereof.
 36. The method of claim 15, wherein said adenoviral vector is further deleted for the adenoviral E3-region or a functional part thereof.
 37. The method of claim 35, wherein said adenoviral vector is further deleted for the adenoviral E4-region or a functional part thereof.
 38. The method of claim 36, wherein said adenoviral vector is further deleted for the adenoviral E4-region or a functional part thereof.
 39. The method of claim 33, wherein said promoter is repressed by an adenoviral E1 gene product.
 40. The method of claim 34, wherein said promoter is repressed by an adenoviral E1 gene product.
 41. The method of claim 39, wherein said promoter is an AP1 dependent promoter.
 42. The method of claim 40, wherein said promoter is an AP1 dependent promoter.
 43. A method according to claim 40, wherein said adenoviral vector is packaged into an adenoviral capsid.
 44. The method of claim 1 wherein said function comprises a biological activity.
 45. The method of claim 44 wherein said biological activity is selected from the group consisting of altered viability, morphologic changes, apoptosis, DNA synthesis, tumorigenesis, disease or drug susceptibility, chemical responsiveness, chemical secretion and protein expression.
 46. The method of claim 1 wherein said unique expressible nucleic acid is derived from the group consisting of mammals, fish, nemotodes, insects, yeasts, fungi, bacteria and plants.
 47. The method of claim 46 wherein said library of unique nucleic acid is derived from human placenta mRNA.
 48. The method of claim 46 wherein said library of unique nucleic acid is derived from zebrafish mRNAs.
 49. The method of claim 46 wherein said host cells are present in zebrafish embryos.
 50. The method of claim 46 wherein said host cells are present in zebrafish adults.
 51. A method for determining the function of a unique nucleic acid present in a library, said method comprising: (a) growing a plurality of cell cultures containing at least one cell, said one cell expressing adenoviral sequence consisting essentially of E1-region sequences and expressing one or more functional gene products encoded by at least one adenoviral region selected from an E2A region and an E4 region; and (b) transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one cell in each of said plurality of cell cultures with i) an adapter plasmid comprising adenoviral sequence coding, in operable configuration, for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sequences sufficient to allow for homologous recombination with a first recombinant nucleic acid, and not coding for E1 region sequences which overlap with E1 region sequences in said at least one cell, for E1 region sequences which overlap with E1 region sequences in a first recombinant nucleic acid, for E2B region sequences other than essential E2B sequences, for E2A region sequences, for E3 region sequences and for E4 region sequences, and further comprises a unique nucleic acid sequence and promoter operatively linked to said unique nucleic acid sequence; and ii) a first recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for a functional adenoviral Inverted Terminal Repeat and for sequences sufficient for replication in said at least one cell, but not comprising adenoviral E1 region sequences which overlap with E1 sequences in said at least one cell, and not comprising E2A region sequences or E4 region sequences expressed in said plurality of cells which would otherwise lead to production of replication competent adenovirus wherein said first recombinant nucleic acid has sufficient overlap with said adapter plasmid to provide for homologous recombination resulting in production of recombinant adenoviral vectors in said at least one cell; (c) incubating said plurality of cells under conditions which result in the lysis of said plurality of cells facilitating the release of said recombinant adenoviral vectors containing said unique nucleic acid; and (d) transferring an aliquot of said adenoviral vectors into a corresponding plurality of host cell cultures consisting of cells in which said vectors do not replicate, but in which said nucleic acids are expressible; (e) incubating said host cells to allow expression of the product of said nucleic acid; and (f) observing said host cell for changes in said host cell.
 52. A method according to claim 51, further comprising (g) assigning a function to said nucleic acids, for which the expressed product thereof results in observed changes in said host cells.
 53. A method according to claim 52, wherein said biological function comprises apoptosis, DNA synthesis, tumorigenesis, disease or drug susceptibility, chemical responsiveness, chemical secretion, protein expression, cell differentiation, proliferation, drug resistance, capillary formation, or cell migration.
 54. A method according to claim 52, wherein said observing uses an assay selected from the group consisting of a beta-galactosidase assay, hIL3 assay, Luciferase assay, ceNOS assay, GLVR2 assay and EGFP assay.
 55. A method according to claim 52 wherein said zebrafish host cells comprise embryos.
 56. The method of claim 52 said zebrafish host cells comprise adult cells. 